Specific sequence motifs direct the oxygenation and chlorination of tryptophan by myeloperoxidase

Most studies of protein oxidation have typically focused on the reactivity of single amino acid side chains while ignoring the potential importance of adjacent sequences in directing the reaction pathway. We previously showed that hypochlorous acid (HOCl), a specific product of myeloperoxidase, inactivates matrilysin by modifying adjacent tryptophan and glycine (WG) residues in the catalytic domain. Here, we use model peptides that mimic the region of matrilysin involved in this reaction, VVWGTA, VVWATA, and the library VVWXTA, to determine whether specific sequence motifs are targeted for chlorination or oxygenation by myeloperoxidase. Our results demonstrate that HOCl generated by myeloperoxidase or activated neutrophils converts the peptide VVWGTA to a chlorinated product, WG+32(Cl). Tandem mass spectrometry in concert with high resolution 1H and two-dimensional NMR analysis revealed that the modification required cross-linking of the tryptophan to the amide of glycine followed by chlorination of the indole ring of tryptophan. In contrast, when glycine in the peptide was replaced with alanine, the major products were mono- and dioxygenated tryptophan residues. When the peptide library VVWXTA (where X represents all 20 common amino acids) was exposed to HOCl, only WG produced a high yield of the chloroindolenine derivative. However, when glycine was replaced by other amino acids, oxygenated tryptophan derivatives were the major products. Our observations indicate that WG may represent a specific sequence motif in proteins that is targeted for chlorination by myeloperoxidase.     

Products of the reaction of HOCl with tryptophan protect LDL from atherogenic modification

Hypochlorite (HOCl) attacks amino acid residues in LDL making the particle atherogenic. Tryptophan is prone to free radical reactions and modification by HOCl. We hypothesized, that free tryptophan may quench the HOCl attack therefore protecting LDL. Free tryptophan inhibits LDL apoprotein modification and lipid oxidation. Tryptophan-HOCl metabolites associate with LDL reducing its oxidizability initiated by endothelial cells, Cu(2+) and peroxyl radicals. One tryptophan-HOCl metabolite was identified as 4-methyl-carbostyril which showed antioxidative activity when present during Cu(2+) mediated lipid oxidation, but did not associate with LDL. Indole-3-acetaldehyde, a decomposition product of tryptophan chloramine (the product of the tryptophan-HOCl reaction) was found to associate with LDL increasing its resistance to oxidation. Myeloperoxidase treatment of LDL in the presence of chloride, H(2)O(2) and tryptophan protected the lipoprotein from subsequent cell-mediated oxidation. We conclude that, in vivo, the activated myeloperoxidase system can generate antioxidative metabolites from tryptophan by the reaction of hypochlorite with this essential amino acid.     

Chemiluminescence associated with amino acid oxidation mediated by hypochlorous acid

Although most amino acids readily react with hypochlorous acid (HOCl), only the reaction involving tryptophan (Trp) produces a measurable chemiluminescence (CL). Most of this luminescence takes place after total consumption of HOCl when the process is carried out in an excess of Trp. The quantum yield of the process is relatively low (2 × 10^?8 Einstein/mol HOCl reacted). The luminescence is attributed to free radical‐mediated secondary reactions of the initially produced chloramines. This is supported by experiments showing that the chloramines produced when HOCl reacts with alanine are able to induce Trp chemiluminescence, and that this luminescence is partially quenched by free radical scavengers. The spectral changes and the effect of pH upon the observed luminescence are compatible with light emission from products produced in the free radical oxidation of Trp. Copyright © 2002 John Wiley & Sons, Ltd.     

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。     

Physiological evidence for differently regulated tryptophan-dependent pathways for indole-3-acetic acid synthesis in Azospirillum brasilense

Disruption of ipdC, a gene involved in indole-3-acetic acid (IAA) production by the indole pyruvate pathway in Azospirillum brasilense Sp7, resulted in a mutant strain that was not impaired in IAA production with lactate or pyruvate as the carbon source. A tryptophan auxotroph that is unable to convert indole to tryptophan produced IAA if tryptophan was present but did not synthesise IAA from indole. Similar results were obtained for a mutant strain with additional mutations in the genes ipdC and trpD. This suggests the existence of an alternative Trp-dependent route for IAA synthesis. On gluconate as a carbon source, IAA production by the ipdC mutant was inhibited, suggesting that the alternative route is regulated by catabolite repression. Using permeabilised cells we observed the enzymatic conversion of tryptamine and indole-3-acetonitrile to IAA, both in the wild-type and in the ipdC mutant. IAA production from tryptamine was strongly decreased when gluconate was the carbon source.     

Endogenous and dietary indoles: a class of antioxidants and radical scavengers in the ABTS assay

Indoles are very common in the body and diet and participate in many biochemical processes. A total of twenty-nine indoles and analogs were examined for their properties as antioxidants and radical scavengers against 2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) ABTS*+ radical cation. With only a few exceptions, indoles reacted nonspecifically and quenched this radical at physiological pH affording ABTS. Indoleamines like tryptamine, serotonin and methoxytryptamine, neurohormones (melatonin), phytohormones (indoleacetic acid and indolepropionic acid), indoleamino acids like L-tryptophan and derivatives (N-acetyltryptophan, L-abrine, tryptophan ethyl ester), indolealcohols (tryptophol and indole-3-carbinol), short peptides containing tryptophan, and tetrahydro-beta-carboline (pyridoindole) alkaloids like the pineal gland compound pinoline, acted as radical scavengers and antioxidants in an ABTS assay-measuring total antioxidant activity. Their trolox equivalent antioxidant capacity (TEAC) values ranged from 0.66 to 3.9 mM, usually higher than that for Trolox and ascorbic acid (1 mM). The highest antioxidant values were determined for melatonin, 5-hydroxytryptophan, trp-trp and 5-methoxytryptamine. Active indole compounds were consumed during the reaction with ABTS*+ and some tetrahydropyrido indoles (e.g. harmaline and 1-methyl-1,2,3,4-tetrahydro-beta-carboline-3-carboxylic acid ethyl ester) afforded the corresponding fully aromatic beta-carbolines (pyridoindoles), that did not scavenge ABTS*+. Radical scavenger activity of indoles against ABTS*+ was higher at physiological pH than at low pH. These results point out to structural compounds with an indole moiety as a class of radical scavengers and antioxidants. This activity could be of biological significance given the physiological concentrations and body distribution of some indoles.     

Inhibition of hypochlorous acid-mediated reactions by desferrioxamine. Implications for the mechanism of cellular injury by neutrophils

Inhibition of free radical mechanisms by desferrioxamine, an iron chelator, is often thought to be a good indicator of iron-catalyzed hydroxyl radical (OH.) production. The specificity of desferrioxamine is critical for such identification. This study was undertaken to determine whether desferrioxamine could prevent the in vitro cytotoxic reactions of hypochlorous acid (HOCl), a major neutrophil-derived oxidant. Red blood cells were used as a target for HOCl, and cell lysis and haemoglobin oxidation were measured. Desferrioxamine, and its iron-chelated form, ferrioxamine, were shown to prevent both effects of HOCl. However, desferrioxamine was 6 to 8 times more efficient than either ferrioxamine or taurine, another amine which prevents HOCl-mediated cell lysis, in preventing both lysis and Hb oxidation. After reaction with HOCl, ferrioxamine and taurine retained almost all the oxidizing equivalents as long-lived chloramine. However, with desferrioxamine less than half the oxidizing equivalents were recovered as chloramines indicating that sites other than the terminal amine reacted with HOCl. The chloramines formed were able to oxidize molecules in solution, but being hydrophilic they were confined to the extracellular medium and cell lysis did not occur. The results indicate that scavenging of HOCl could be a factor in the inhibition by desferrioxamine of neutrophil-mediated cell lysis in vitro.     

Creation of a Metabolic Sink for Tryptophan Alters the Phenylpropanoid Pathway and the Susceptibility of Potato to Phytophthora infestans

The creation of artificial metabolic sinks in plants by genetic engineering of key branch points may have serious consequences for the metabolic pathways being modified. The introduction into potato of a gene encoding tryptophan decarboxylase (TDC) isolated from Catharanthus roseus drastically altered the balance of key substrate and product pools involved in the shikimate and phenylpropanoid pathways. Transgenic potato tubers expressing the TDC gene accumulated tryptamine, the immediate decarboxylation product of the TDC reaction. The redirection of tryptophan into tryptamine also resulted in a dramatic decrease in the levels of tryptophan, phenylalanine, and phenylalanine-derived phenolic compounds in transgenic tubers compared with nontransformed controls. In particular, wound-induced accumulation of chlorogenic acid, the major soluble phenolic ester in potato tubers, was found to be two- to threefold lower in transgenic tubers. Thus, the synthesis of polyphenolic compounds, such as lignin, was reduced due to the limited availability of phenolic monomers. Treatment of tuber discs with arachidonic acid, an elicitor of the defense response, led to a dramatic accumulation of soluble and cell wall-bound phenolics in tubers of untransformed potato plants but not in transgenic tubers. The transgenic tubers were also more susceptible to infection after inoculation with zoospores of Phytophthora infestans, which could be attributed to the modified cell wall of these plants. This study provides strong evidence that the synthesis and accumulation of phenolic compounds, including lignin, could be regulated by altering substrate availability through the introduction of a single gene outside the pathway involved in substrate supply. This study also indicates that phenolics, such as chlorogenic acid, play a critical role in defense responses of plants to fungal attack.     

The pathway of auxin biosynthesis in plants

The plant hormone auxin, which is predominantly represented by indole-3-acetic acid (IAA), is involved in the regulation of plant growth and development. Although IAA was the first plant hormone identified, the biosynthetic pathway at the genetic level has remained unclear. Two major pathways for IAA biosynthesis have been proposed: the tryptophan (Trp)-independent and Trp-dependent pathways. In Trp-dependent IAA biosynthesis, four pathways have been postulated in plants: (i) the indole-3-acetamide (IAM) pathway; (ii) the indole-3-pyruvic acid (IPA) pathway; (iii) the tryptamine (TAM) pathway; and (iv) the indole-3-acetaldoxime (IAOX) pathway. Although different plant species may have unique strategies and modifications to optimize their metabolic pathways, plants would be expected to share evolutionarily conserved core mechanisms for auxin biosynthesis because IAA is a fundamental substance in the plant life cycle. In this review, the genes now known to be involved in auxin biosynthesis are summarized and the major IAA biosynthetic pathway distributed widely in the plant kingdom is discussed on the basis of biochemical and molecular biological findings and bioinformatics studies. Based on evolutionarily conserved core mechanisms, it is thought that the pathway via IAM or IPA is the major route(s) to IAA in plants.     

Naphthoindole-based analogues of tryptophan and tryptamine: synthesis and cytotoxic properties

The efficacy of anthracycline based anticancer drugs is limited by pleiotropic drug resistance of tumor cells. Aiming at the design of anthracyclinone congeners capable of circumventing drug resistance, we synthesized naphthoindole containing derivatives of tryptophan and tryptamine. In doing so we adapted the traditional, gramine based approach for tryptophan and tryptamine synthesis. The most potent new compound, 3-(2-aminoethyl)-4,11-dihydroxynaphtho[2,3-f]indole-5,10-dione (16), was equally cytotoxic (IC(50) within low micromolar concentrations) for human K562 leukemia and HCT116 colon carcinoma cell lines and their isogenic sublines with genetically defined determinants of altered drug response, that is, the expression of the multidrug transporter P-glycoprotein and loss of pro-apoptotic p53. Each of these mechanisms conferred resistance to the reference drug adriamycin. In contrast, naphthotryptamine 16, although less potent than adriamycin, was equally toxic for wild type cell lines and drug resistant counterparts. Moreover, at 3-5 microM 16 inhibited topoisomerase I in vitro. Thus, our novel naphthoindole based derivative of tryptamine gained new activities important for anticancer therapy, namely, suppression of topoisomerase I and the ability to overcome resistance mediated by P-glycoprotein expression and p53 dysfunction.     

Hypochlorous acid stress in Escherichia coli: resistance, DNA damage, and comparison with hydrogen peroxide stress.

We have investigated the mechanisms of killing of Escherichia coli by HOCl by identifying protective functions. HOCl challenges were performed on cultures arrested in stationary phase and in exponential phase. Resistance to HOCl in both cases was largely mediated by genes involved in resistance to hydrogen peroxide (H2O2). In stationary phase, a mutation in rpoS, which controls the expression of starvation genes including those which protect against oxidative stress, renders the cells hypersensitive to killing by HOCl. RpoS-regulated genes responsible for this sensitivity were dps, which encodes a DNA-binding protein, and, to a lesser extent, katE and katG, encoding catalases; all three are involved in resistance to H2O2. In exponential phase, induction of the oxyR regulon, an adaptive response to H2O2, protected against HOCl exposure, and the oxyR2 constitutive mutant is more resistant than the wild-type strain. The genes involved in this oxyR-dependent resistance have not yet been identified, but they differ from those primarily involved in resistance to H2O2, including katG, ahp, and dps. Pretreatment with HOCl conferred resistance to H2O2 in an OxyR-independent manner, suggesting a specific adaptive response to HOCl. fur mutants, which have an intracellular iron overload, were more sensitive to HOCl, supporting the generation of hydroxyl radicals upon HOCl exposure via a Fenton-type reaction. Mutations in recombinational repair genes (recA or recB) increased sensitivity to HOCl, indicative of DNA strand breaks. Sensitivity was visible in the wild type only at concentrations above 0.6 mg/liter, but it was observed at much lower concentrations in dps recA mutants.     

Determination of metabolic rate-limitations by precursor feeding in Catharanthus roseus hairy root cultures

Precursors from the terpenoid and tryptophan branches were fed to Catharanthus roseus to determine which of the two branches limits metabolic flux to indole alkaloids. The feeding of tryptophan at 17 days of the culture cycle produced auxin-like effects. Addition of low levels of auxin or tryptophan resulted in significant increases in flux to the indole alkaloids. Conversely, feeding higher levels of auxin or tryptophan resulted in increased branching and thickening of the hairy root cultures. A dramatic reduction in flux to the alkaloids was also observed. However, feeding tryptamine or terpenoid precursors had no effect. Therefore, neither pathway tested revealed to be rate-limiting during the late growth phase. Feeding of either geraniol, 10-hydroxygeraniol, or loganin at 21 days each resulted in significant increases in the accumulation of tabersonine. The addition of tryptophan or tryptamine had no effect during the stationary phase of the growth cycle. Thus, during the early stationary phase of growth the terpenoid pathway appears to be rate-limiting. Combined elicitation with jasmonic acid and feeding either loganin or tryptamine did not further enhance the accumulation of indole alkaloids.     

Chlorination by a long-lived intermediate in the mechanism of flavin-dependent halogenases

The flavin-dependent halogenase RebH catalyzes the formation of 7-chlorotryptophan as the initial step in the biosynthesis of antitumor agent rebeccamycin. The reaction of FADH2, Cl-, and O2 in the active site generates the powerful oxidant HOCl, which was presumed to carry out the chlorination reaction. Herein, we demonstrate the formation of a long-lived chlorinating intermediate (t1/2 = 63 h at 4 degrees C) when RebH, FADH2, Cl-, and O2 react in the absence of substrate tryptophan. This intermediate remained on the enzyme after removal of FAD and transferred chlorine to tryptophan with kinetically competent rates. The identity of this intermediate is suggested by the X-ray crystal structure of RebH, which revealed an active site Lys79 located in a central position between flavin and tryptophan binding sites and just 4.1 A above C7 of tryptophan. The chlorinating species is proposed to be a Lys-epsilonNH-Cl (lysine chloramine) from reaction of enzyme-generated HOCl with the active site Lys79. This covalent enzyme chloramine likely plays a key role in directing regiospecific chlorination of substrate in this important class of biosynthetic enzymes.     

Biotransformation of indole derivatives by mycelial cultures

Biotransformation of tryptophan to tryptamine and 3-methyl-indole by Psilocybe coprophila was performed. On the other hand, Aspergillus niger was able to transform tryptophan to 5-hydroxy-tryptophan. P. coprophila biotransformed 5-hydroxy-tryptophan to 5-hydroxytryptamine. These results prove once more that fungi are good tools to establish hydroxyindole derivatives.     

Effects of Light Quality on Induction of Tryptamine-mediated Resistance in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

The effective wavelength of light on Sekiguchi lesion formation, tryptamine accumulation, enzyme activities and hydrogen peroxide (H₂O₂) generation involved in light‐induced resistance of rice (cv. Sekiguchi‐asahi) infected with Magnaporthe grisea was determined by using band‐pass filters (BPF). When the M. grisea‐inoculated leaves were irradiated by BPF‐filtered lights from daylight fluorescent lamps, Sekiguchi lesion development was enhanced by increasing the wavelength of visible light. The longer the radiation wavelength, the greater the tryptamine accumulation, the tryptophan decarboxylase and monoamine oxidase activities, and the H₂O₂ generation. By contrast, the longer the radiation wavelength of radiation, the greater the inhibition of catalase (CAT) activity. Among the visible lights, red light was the most effective for tryptamine pathway activation and CAT inhibition involved in light‐induced resistance.     

Impact of auxin-compounds produced by the antagonistic fungus Pythium oligandrum or the minor pathogen Pythium group F on plant growth

Plant growth promotion induced by the antagonistic fungus, Pythium oligandrum, is the result of a complex interaction which includes an indirect effect through control of pathogens in the rhizosphere and/or a direct one mediated by plant-induced resistance. The present study shows an increased plant growth associated with direct interaction between P. oligandrum and roots, which is mediated by a fungus-produced auxin compound, tryptamine (TNH₂). In vitro experiments provided evidence that P. oligandrum metabolised specifically indole derivatives, such as tryptophan and indole-3-acetaldehyde, to produce THN₂ through the tryptamine pathway. When P. oligandrum grew in sterile root exudates, it also produced an auxin-like compound. Additional experiments on P. oligandrum–root interaction showed that, in amended nutrient solution of plants, the antagonist metabolised Trp into TNH₂ and that root absorption of this newly formed auxin-compound in appropriate concentrations was associated with enhancement of plant growth. This phenomenon was observed only when nutrient solution was amended with low tryptophan (Trp) concentrations, i.e. 0.05 and 0.1 mM; higher concentration (0.5 and 1 mM Trp) induced abnormal root development. Similar experiments were performed with Pythium group F, a minor pathogen known for its ability to produce auxin-compounds through the tryptamine pathway. In this case, irregular root development was always noticed with all Trp concentrations added to the nutrient solution of plants. Moreover, Pythium group F colonization of roots was associated with leakage of auxin-compounds in the nutrient solution. Our results, therefore, highlight that the production of similar auxin-compounds by two Pythium species has contrary effects on plant development.     

Oxidation of deseferrioxamine to nitroxide free radical by activated human neutrophils

Human neutrophils activatd by PMA were found to induced the formation of a nitroxide radical from DFO. The presence of SOD was necessary to permit the formation of the DFO radical. The inactive phorbol ester did not induce DFO radical, and _sphinganine suppressed the radical produced by the active phorbol ester. Other cell stimuli (Zymocel and the chemotactic peptide) also induced the formation of the DFO radical, although radical concentration was very much lower than with PMA. Participation of ^.NO, ^,OH or ¹O₂ was ruled out by the inability of N^G-methyl-L-arginine, N^G-nitro-L-arginine, DMSO, mannitol, histidine, and methionine to inhibit the formation of DFO radical produced by PMA-activated cells. Furthermore, PMA-activated cells dod not produce detectable levels of NO₂⁻, as a stable oxidation product of ^.NO, and D₂, which enhances the lifetime of singlet oxygen, did not modify the intensity or the lifetime of DFO radical. The involvement of cell MPO was suggested by the inhibition of the DFO radical observed after treatment with catalase or with antihuman MPO antibodies. Also, HOCI was found to induce the DFO radical in cell-free reactions, but our data indicate that the reaction leading to DFO radical formation by neutrophils involves the reduction of MPO compound II back to active enzyme (ferric-MPO). Anti-inflammatory drugs strongly increased the DFO radical produced by activated neutrophils. On the contrary, none of these drugs was able to increase the DFO radical produced by HOCl. Histidine and methionine that inhibited the DFO radical intensity in cell-free reactions, were shown to act directly onm HOCl. Experiments with MPO-H₂O₂ in SOD- and Cl⁻-free conditions showed the formation of DFO radical and confirmed the hypothesis of the involvement of compound II. The conversion of compound II to ferric MPO by DFO optimized the enzymatic activity of neurophils, and in the presence of monochlorodimedon (compound II promoting agent) we measured an increased HOCl production. When DFO was modified by conjugation with hydroxyethyl starch, it lost the ability to produce the radical either by neutrophils or by MPO-H₂O₂ and did not increase HOCl production. The inability of these DFO derivatives to produce potentially toxic species migh explain their reported lower toxicity in vivo.     

Effect of Photosynthetic Inhibitors on Induction of Tryptamine Pathway-mediated Resistance in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

Rice cv. Sekiguchi‐asahi was identified as a lesion mimic mutant from cv. Asahi. This mutant showed light‐enhanced resistance to Magnaporthe grisea infection, inducing Sekiguchi lesion (sl) formation and tryptamine accumulation. To elucidate the role of chloroplasts on the activation of tryptamine pathway, the effect of some photosynthetic inhibitors on light‐dependant Sekiguchi lesion formation, tryptamine accumulation, and enzyme activities was investigated. When detached leaf blades which had been pretreated with Linuron, 1, 10‐Phenanthroline or Poly‐l ‐lysine were inoculated with M. grisea, respectively, Sekiguchi lesion formation and typtamine accumulation were suppressed even under light, suppressing gene expression of tryptophan decarboxylase and monoamine oxidase activity. On the other hand, catalase activity was significantly inhibited in control leaves infected with M. grisea under light, but kept at high level in chemical‐pretreated leaves. These results suggested that photosynthetic activity in chloroplasts was involved in inhibition of antioxidant sysytem and activation of tryptamine pathway responsible for light‐enhanced resistance in sl mutant to M. grisea infection.     

Tryptophan Decarboxylase,Tryptamine, and Reproduction of the Whitefly

Tryptophan decarboxylase (TDC) from Catharanthus roseus (periwinkle) converts tryptophan to the indole-alkaloid tryptamine. When the TDC gene was expressed in transgenic tobacco, the 55-kD TDC enzyme and tryptamine accumulated. Bemisia tabaci (sweetpotato whitefly) reproduction on transgenic plants decreased up to 97% relative to controls. Production of tryptamine, its derivatives, or other products resulting from TDC activity may discourage whitefly reproduction and provide a single-gene-based plant protection strategy.