Tryptophan Side Chain Oxidase Research Articles

Biosynthetic Pathways and Functions of Indole-3-Acetic Acid in Microorganisms.

Indole-3-acetic acid (IAA) belongs to the family of auxin indole derivatives. IAA regulates almost all aspects of plant growth and development, and is one of the most important plant hormones. In microorganisms too, IAA plays an important role in growth, development, and even plant interaction. Therefore, mechanism studies on the biosynthesis and functions of IAA in microorganisms can promote the production and utilization of IAA in agriculture. This mini-review mainly summarizes the biosynthesis pathways that have been reported in microorganisms, including the indole-3-acetamide pathway, indole-3-pyruvate pathway, tryptamine pathway, indole-3-acetonitrile pathway, tryptophan side chain oxidase pathway, and non-tryptophan dependent pathway. Some pathways interact with each other through common key genes to constitute a network of IAA biosynthesis. In addition, functional studies of IAA in microorganisms, divided into three categories, have also been summarized: the effects on microorganisms, the virulence on plants, and the beneficial impacts on plants.

Tryptophan Side-Chain Oxidase (TSO) Degrades L-Tryptophan, a Possible New Cancer Therapy

Not all cancer therapeutic strategies known to date are adequate for all cancer types and patients. Many of them are followed by a high rate of side effects and complications. L-Tryptophan metabolism plays a key role in organism development. As well as in the occurrence and development of tumors. By degrading certain amino acids, tumor growth can be limited while maintaining the body ?s normal nutritional requirements. L-tryptophan depletion bioreactor is described as a possible new therapeutic strategy of cancer therapy. L-tryptophan is an essential amino acid, which has been recognized as an important cancer nutrient and its removal can lead to destruction of the tumor. Normal human cells or cancer cells cannot synthesize L-tryptophan and therefor tumor resistance is unlikely to develop. L-tryptophan is also a constituent for different bio-molecules such as Serotonin, Melatonin, and is needed for other synthesis processes in the cell growth. A column, which contained tryptophan side-chain oxidase (TSO) as a bioreactor, was integrated in a therapeutic plasma exchange unit, and tested it in different animals and cancer cell lines. Breast cancer, medulloblastoma, and hepatocellular carcinoma showed greatest efficacy of L-tryptophan degrading. TSO is developed to treat cancer diseases successfully, and has low side effects. A combination of L-tryptophan depletion with all available cancer therapies is possible.

Tryptophan Side-Chain Oxidase Enzyme Suppresses Hepatocellular Carcinoma Growth through Degradation of Tryptophan.

Tryptophan metabolism plays a role in the occurrence and development of hepatocellular carcinoma cells. By degrading certain amino acids, tumor growth can be limited while maintaining the body’s normal nutritional requirements. Tryptophan side-chain oxidase (TSO) enzyme can degrade tryptophan, and its inhibitory effect on hepatocellular carcinoma cells is worthy of further study. To investigate the degradation effect on tryptophan, TSO was isolated and purified from qq Pseudomonas. The reaction products were identified with high performance liquid chromatography (HPLC) and high-performance liquid chromatography tandem mass spectrometry (HPLC-MS). De novo sequencing provided the complete amino acid sequence of TSO. The results of CCK-8, colony formation, transwell, and qPCR confirmed that TSO had inhibitory effects on the proliferation and migration of HCCLM3 (human hepatocarcinoma cell line) and HepG2 cells. The results of flow cytometry confirmed its apoptotic activity. In animal experiments, we found that the tumor-suppressive effect was better in the oncotherapy group than the intraperitoneal injection group. The results of immunohistochemistry also suggested that TSO could inhibit proliferation and promote apoptosis. In conclusion, a specific enzyme that can degrade tryptophan and inhibit the growth of hepatoma cells was authenticated, and its basic information was obtained by extraction/purification and amino acid sequencing.

Unusual reactivity of β-hydroxy-serotonin, a forgotten serotonin derivative.

Serotonin (5-HT) is a well-known biogenic amine which regulates mood, sleep, and is involved in muscle contraction and blood coagulation. Based on an analogy to norepinephrine, a β-hydroxylated derivative of dopamine which has diverse physiological functions, beta-hydroxy-serotonin (β-OH-5-HT) originally encouraged interest as a potential pharmacological agent. Four decades ago, its organic synthesis was attempted. However, due to difficulties with the synthesis and the compound's instability, rigorous identification and characterization of β-OH-5-HT proved evasive. Here, we successfully synthesized β-OH-5-HT from 5-HT using a Pseudomonas enzyme, tryptophan side chain oxidase type I (TSOI), and we determined the structure by 2D-NMR and characterized β-OH-5-HT in detail. The CD spectra showed no optical activity, suggesting a racemic mixture. To separate DL-β-OH-5-HT, we synthesized L-Ala-5-HT and derivatized it into erythro- and threo-L-Ala-β-OH-5-HT with TSOI. Interestingly, both isolated fractions returned to a diastereoisomeric mixture within two hours at pH 5.0. Later, we found that, under acidic conditions, β-OH-5-HT readily reacted with nucleophiles like alcohols or thiols, yielding a variety of DL-β-substituted-5-HT. The unusual properties of β-OH-5-HT might be attributed to the unique nature of a β-hydroxyl group adjacent to an indole ring and amino group. The mechanism for the rapid racemization of β-OH-5-HT is discussed.

Novel biochemical manipulation of brain serotonin reveals a role of serotonin in the circadian rhythm of sleep–wake cycles

Serotonin (5-HT) neurons have been implicated in the modulation of many physiological functions, including mood regulation, feeding, and sleep. Impaired or altered 5-HT neurotransmission appears to be involved in depression and anxiety symptoms, as well as in sleep disorders. To investigate brain 5-HT functions in sleep, we induced 5-HT deficiency through acute tryptophan depletion in rats by intraperitoneally injecting a tryptophan-degrading enzyme called tryptophan side chain oxidase I (TSOI). After the administration of TSOI (20 units), plasma tryptophan levels selectively decreased to 1-2% of those of controls within 2 h, remained under 1% for 12-24 h, and then recovered between 72 and 96 h. Following plasma tryptophan levels, brain 5-HT levels decreased to ∼30% of the control level after 6 h, remained at this low level for 20-30 h, and returned to normal after 72 h. In contrast, brain norepinephreine and dopamine levels remained unchanged. After TSOI injection, the circadian rhythms of the sleep-wake cycle and locomotive activity were lost and broken into minute(s) ultradian alternations. The hourly slow-wave sleep (SWS) time significantly increased at night, but decreased during the day, whereas rapid eye movement sleep was significantly reduced during the day. However, daily total (cumulative) SWS time was retained at the normal level. As brain 5-HT levels gradually recovered 48 h after TSOI injection, the circadian rhythms of sleep-wake cycles and locomotive activity returned to normal. Our results suggest that 5-HT with a rapid turnover rate plays an important role in the circadian rhythm of sleep-wake cycles.

Statistical optimization for improved indole-3-acetic acid (iaa) production by Pseudomonas aeruginosa and demonstration of enhanced plant growth promotion

rd block, Bangalore -560011, India. * Abstract The present study was undertaken to study the statistical optimization of medium components for improved Indole-3- acetic acid (IAA) production by Pseudomo- nas aeruginosa. Colorimetric analysis showed maximum IAA (132 µg mL -1 ) in the medium supplemented with tryptophan (0.5 g L -1 ). The maximum production was achieved after 96 h of incubation. Yeast extract, tryptophan and EDTA were identi- fied as significant components influencing IAA production by Pseudomonas aeru- ginosa, using the Plackett- Burman method. The statistical optimization approach led to the production of 318 µg mL -1 of IAA within 24 h of incubation. Statistical approach was found to be very effective in optimizing the medium components in a manageable number of experimental runs with overall 2.4 fold increase in IAA pro- duction. TLC and GC- MS analysis further confirmed the IAA production in the cell filtrates of the strain. GC-MS analysis and tryptophan side chain oxidase confirmed the existence of atleast 2 possible pathways for IAA by this strain. Inoculation of P. aeruginosa culture filtrate enhanced seed germination (82.4%) and increase in root length and shoot length (~ 2.6 and ~ 1.1 folds over the control) of cowpea seeds over the control treatment under pot culture conditions.

Aerobic methylobacteria are capable of synthesizing auxins

Obligately and facultatively methylotrophic bacteria with different pathways of C1 metabolism were found to be able to produce auxins, particularly indole-3-acetic acid (IAA), in amounts of 3-100 micrograms/ml. Indole-3-pyruvic acid and indole-3-acetamide were detected only in methylobacteria with the serine pathway of C1 metabolism, Methylobacterium mesophilicum and Aminobacter aminovorans. The production of auxins by methylobacteria was stimulated by the addition of tryptophan to the growth medium and was inhibited by ammonium ions. The methylobacteria under study lacked tryptophan decarboxylase and tryptophan side-chain oxidase. At the same time, they were found to contain several aminotransferases. IAA is presumably synthesized by methylobacteria through indole-3-pyruvic acid.

Enhanced production of indole-3-acetic acid by a genetically modified strain of Pseudomonas fluorescens CHA0 affects root growth of cucumber, but does not improve protection of the plant against Pythium root rot

The biocontrol strain CHA0 of Pseudomonas fluorescens produces small amounts of indole-3-acetic acid via the tryptophan side chain oxidase and the tryptophan transaminase pathways. A recombinant plasmid (pME3468) expressing the tryptophan monooxygenase pathway was introduced into strain CHA0; this resulted in elevated synthesis of indole-3-acetic acid in vitro, especially after addition of l-tryptophan. In natural soil, strain CHA0/pME3468 increased fresh root weight of cucumber by 17–36%, compared to the effect of strain CHA0; root colonization was about 10 6 cells per g of root. However, both strains gave similar protection of cucumber against Pythium ultimum. In autoclaved soil, at 6×10 7 cells per g of root, strain CHA0 stimulated growth of roots and shoots, whereas strain CHA0/pME3468 caused root stunting and strong reduction of plant weight. These results are in agreement with the known effects of exogenous indole-3-acetic acid on plant roots and suggest that in the system examined, indole-3-acetic acid does not contribute to the biocontrol properties of strain CHA0.

Enhanced production of indole-3-acetic acid by a genetically modified strain of Pseudomonas fluorescens CHA0 affects root growth of cucumber, but does not improve protection of the plant against Pythium root rot

The biocontrol strain CHA0 of Pseudomonas fluorescens produces small amounts of indole-3-acetic acid via the tryptophan side chain oxidase and the tryptophan transaminase pathways. A recombinant plasmid (pME3468) expressing the tryptophan monooxygenase pathway was introduced into strain CHA0; this resulted in elevated synthesis of indole-3-acetic acid in vitro, especially after addition of l-tryptophan. In natural soil, strain CHA0/pME3468 increased fresh root weight of cucumber by 17–36%, compared to the effect of strain CHA0; root colonization was about 106 cells per g of root. However, both strains gave similar protection of cucumber against Pythium ultimum. In autoclaved soil, at 6×107 cells per g of root, strain CHA0 stimulated growth of roots and shoots, whereas strain CHA0/pME3468 caused root stunting and strong reduction of plant weight. These results are in agreement with the known effects of exogenous indole-3-acetic acid on plant roots and suggest that in the system examined, indole-3-acetic acid does not contribute to the biocontrol properties of strain CHA0.

Depletion of patients' plasma tryptophan using tryptophan side-chain oxidase columns.

The use of the enzyme tryptophan side-chain oxidase, isolated from Pseudomonas XA, was explored in 3 patients with refractory acute lymphocytic leukemia. Patients were given either a low-tryptophan diet or tryptophan-free hyperalimentation, prior to and during therapy. Their plasma, separated by pheresis, was continuously passed through a tryptophan depletion column containing the immobilized tryptophan side-chain oxidase. Up to 4 plasma volumes were passed through the column daily, 5 days per week for 2-3 weeks, and plasma tryptophan levels, both free and total, were measured by high-performance liquid chromatography. Pre- and postcolumn plasma samples were collected throughout the pheresis procedure. All postcolumn plasma samples had unmeasurable tryptophan levels throughout the treatment period, whereas precolumn samples were always measurable. Generally, tryptophan levels of plasma isolated from peripheral blood decreased after therapy, but rebounded by the next day. The enzyme depletion column reduces circulating plasma tryptophan levels, and its use is well tolerated by patients. However, further development of this method will require study of the effects of diet and of the duration, interval, and frequency of use of this column on therapeutic efficacy. Problems include difficulties with extended diet compliance and apparently intensive mobilization of tryptophan from body stores, which may preclude the clinical application of this enzyme depletion column.

The Synthesis of L-tryptophan Degrading Bioreactors

To design a bioreactor for removing the potential cancer nutrient L-tryptophan from blood, the L-tryptophan degrading enzyme tryptophan side chain oxidase (TSO) was chemically bound to glutaraldehyde activated gamma amino silane silica and to Zetaffinity microcolumns consisting of a glutaraldehyde activated polyacrylic-cellulose copolymer. Five experiments were carried out in sheep and six experiments in rabbits using a closed circuit plasmapheresis bioreactor system. L-tryptophan in sheep was degraded by the silica bioreactor in a single pass to undetectable levels as measured by high performance liquid chromatography (HPLC). Zetaffinity bioreactors degraded L-tryptophan in rabbits to more than 95% in a single pass. Whole blood L-tryptophan levels changed little throughout the experiment indicating a vast extravascular tryptophan pool. Enzyme leakage from the bioreactor was less than 10(-5) IU TSO per ml plasma. The procedures were tolerated well by the animals without any change in vital signs.

Asymmetric tritiation of N‐acetyl α‐β dehydrotryptophanamide

AbstractRhodium‐DiPAMP complex catalyzes the stereoselective addition of two tritium atoms on N‐acetyl −α −β dehydrotryptophanamide. The substrate was prepared by dehydrogenation of N‐acetyltryptophanamide with TSO (Tryptophan‐Side Chain oxidase) from Pseudomonas. Tritiated N‐acetyl‐tryptophanamide was obtained with the theoretical specific radioactivity (58 Ci/mmole). The enantiomeric excess of the L‐diasteroisomer reached 94.4 %. 3NMR spectra indicated the selectivity of 3H‐labelling on the Cα – Cβ double bond. This new approach of tritiation by homogeneous catalysis applied to tryptophanyl‐containing peptides is discussed.

27] Purification and properties of tryptophan side chain oxidase types I and II from pseudomonas

27] Purification and properties of tryptophan side chain oxidase types I and II from pseudomonas

Enzymatic dehydrogenation of tryptophan residues of human globins by tryptophan side chain oxidase II.

Enzymatic dehydrogenation of tryptophan residues in human alpha- and beta-globins has been demonstrated with tryptophan side chain oxidase II, a hemoprotein from Pseudomonas. In 1 M ammonium acetate, pH 4, the enzyme catalyzed the conversion of Trp-14 of alpha-globin to alpha, beta-dehydro-Trp. Under identical conditions, Trp-15 of beta-globin was converted to alpha, beta-dehydro-Trp, whereas Trp-37 was unmodified. No modification was obtained in 0.4% sodium dodecyl sulfate or 8 M urea. The dehydrogenase component of tryptophan side chain oxidase II was also active toward globins in the presence of ferricyanide, whereas tryptophan side chain oxidase I was inert. Upon analyses of the tryptic fragments of the modified beta-globin, two forms of alpha, beta-dehydro-Trp-15 distinguishable by ultraviolet absorption and fluorescence were identified. Each of them rapidly equilibrated with another form on irradiation at 300-380 nm. We tentatively assigned them to two isomers of alpha, beta-dehydro-Trp distinguishable by the configuration around its beta-carbon atom. alpha, beta-Dehydro-Trp in globins and certain polypeptides used herein showed a blue fluorescence at 420 nm. In [alpha, beta-dehydro-Trp 15]beta-globin, the fluorescence was exceptionally intense, the fluorescence yield being comparable to that of NADH. From several lines of evidence, the result was ascribed to the excitation energy transfer from Trp-37 to alpha, beta-dehydro-Trp-15 in the modified beta-globin.

Isolation and characterization of the common intermediate in dichotomous reaction catalyzed by tryptophan side chain oxidase.

Tryptophan side chain oxidase from Pseudomonas catalyzes the pH-dependent dichotomous conversion of tryptophan residues to a,p-didehydro- and p-hydroxytryptophan. When tryptophan side chain oxidase I1 and N-acetyl-L-tryptophanamide, a model substrate, were incubated at 25 “C in 10 mM Tris-HC1, pH 8.0, a 5-min lag was shown for the formation of N-acetyl-a,p-didehydrotryptophanamide, the main product under these conditions. The analysis of the reaction by high performance liquid chromatography showed that an entity distinct from the products accumulated extensively at the initial stage of the reaction and it decayed nonenzymatically with a half-life of 8 min to give the a,pdidehydro derivative. The formation of the #I-hydroxy derivative was favored when the pH was decreased in the latter stage. Nucleophiles such as methylmercaptan, methyl alcohol, pyrrole, cysteine, and glutathione trapped this intermediate to give the corresponding psubstituted derivatives such as N-acetyl-p-methylthioL-tryptophanamide. The unstable intermediate was extractable by methylene chloride, in which the intermediate survived for several days at -20 “C, but in an aqueous solution, it was readily converted to give a,pdidehydro and 8-hydroxy derivatives at ratios prescribed by a pK value of 5.8, or &substituted adducts in the presence of nucleophiles. The dichotomy of the reaction was thus attributed to the nonenzymatic reaction of the common intermediate which was identified by NMR spectrometry and gas chromatography/ mass spectrometry as 5-(3-indolyl)-2-methyl-2-oxazoline-4-carboxamide, a new biochemical entity.

Enzymatic modification of tryptophan residues by tryptophan side chain oxidase I and II from Pseudomonas.

The enzymatic modification of tryptophan residues in Trp-Leu, Leu-Trp, Leu-Trp-Leu, and yeast mating hormone (alpha-factor), a tridecapeptide containing Trp-1 and Trp-3, has been investigated with tryptophan side chain oxidase I crystallized previously from Pseudomonas and tryptophan side chain oxidase II recently isolated from the same organism. With Trp-Leu as substrate, threo- and erythro-beta-hydroxytryptophan residues were identified as primary products and the former perferentially underwent second step dehydrogenation to give beta-ketotryptophan. Free threo- and erythro-beta-hydroxytryptophan, hitherto undescribed tryptophan derivatives, were isolated after enzymatic hydrolysis, identified, and characterized. Leu-alpha, beta-dehydrotryptamine, a new biochemical entity, was a sole main product of Leu-Trp. With Leu-Trp-Leu, diastereoisomeric Leu-beta-hydroxyl-Trp-Leu and Leu-alpha, beta-dehydro-Trp-Leu were formed simultaneously, the pH and ionic strength being determinants of the ratio of these products. Leu-threo-beta-hydroxy-Trp-Leu was also converted to Leu-beta-keto-Trp-Leu, but, distinct from NH2-terminal beta-hydroxytryptophan, both diastereoisomers underwent acid-catalyzed dehydration to give Leu-alpha, beta-dehydro-Trp-Leu. Thus, the mode of modification was shown to be prescribed by the site of tryptophan in peptides and the results were the same with either tryptophan side chain oxidase I or II, except that the latter was almost inactive on Trp-Leu. With any substrate, 3-indolecarboxaldehyde was a sole detectable by-product.

Isolation and reconstitution of two electron transfer components of tryptophan side chain oxidase.

The isolation and reconstitution of two electron transfer components of tryptophan side chain oxidase from Pseudomonas (ATCC 29574) are described. The dehydrogenase component abstracts electrons from the substrate and transfers them to oxidation-reduction dyes such as potassium ferricyanide and 2,6-dichlorophenolindophenol but not to molecular oxygen. It is composed of a single polypeptide chain with a molecular weight of 72,000 and exhibits the absorption spectrum of a reduced b-type cytochrome with maxima at 563, 532, 433, 323, and 278 nm. The oxidase component transfers electrons, derived from the former component, to oxygen, and has a molecular weight of 48,000. The absorption spectrum exhibits broad peaks at 680, 438, and 358 nm, and a peak at 280 nm. On sucrose gradient centrifugation and polyacrylamide gel electrophoresis, these two components are shown to form a molecular complex, which has the reconstituted oxidase activity. The turnover number of the reconstituted enzyme is comparable to that of the native enzyme.

A new metabolic pathway of tryptophan initiated by tryptophan side chain oxidase.

A new metabolic pathway of tryptophan initiated by tryptophan side chain oxidase.

2,5-dihydro-L-phenylalanine: A competitive inhibitor of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase

2,5-Dihydro-L-phenylalanine, an inhibitor of tryptophan 5-monooxygenase, was shown to be a potent inhibitor of indoleamine 2,3-dioxygenase (Ki = 0.23 mM) and tryptophan 2,3-dioxygenase (Ki = 0.70 mM), whereas tryptophan side chain oxidase from Pseudomonas was not inhibited by this compound.

Tryptophan side chain oxidase from Pseudomonas. pH-dependent formation of alpha,beta-didehydro, beta-hydroxy, and beta-keto derivatives of N-acetyltryptophanamide.

Tryptophan side chain oxidase from Pseudomonas. pH-dependent formation of alpha,beta-didehydro, beta-hydroxy, and beta-keto derivatives of N-acetyltryptophanamide.