A kinetic, spectroscopic, and redox study of human tryptophan 2,3-dioxygenase

The family of heme dioxygenases, as exemplified by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase, catalyzes the oxidative cleavage of L-tryptophan to N-formylkynurenine. Here, we describe a bacterial expression system for human tryptophan 2,3-dioxygenase (rhTDO) together with spectroscopic, kinetic, and redox analyses. We find unexpected differences between human tryptophan 2,3-dioxygenase and human indoleamine 2,3-dioxygenase [Chauhan et al. (2008) Biochemistry 47, 4761-4769 ]. Thus, in contrast to indoleamine 2,3-dioxygenase, the catalytic ferrous-oxy complex of rhTDO is not observed, nor does the enzyme discriminate against substrate binding to the ferric derivative. In addition, we show that the rhTDO is also catalytically active in the ferric form. These new findings illustrate that significant mechanistic differences exist across the heme dioxygenase family, and the data are discussed within this broader framework.     

l-Kynurenine Its synthesis and possible regulatory function in brain

One pathway by which tryptophan is metabolized in the brain as well as in the periphery is through cleavage of the indole ring to formylkynurenine and then kynurenine. Indoleamine-2,3-dioxygenase, the enzyme that catalyzes this clavage, and kynurenine are distributed all across the different anatomic regions of brain. Approximately 40% of the kynurenine in brain is synthesized there, the remainder having come from plasma. Tryptophan loading, which has been used both experimentally and therapeutically as a means of increasing tryptophan conversion to serotonin, also increases kynurenine formation in the brain and in the periphery. Because of the formation of kynurenine, which competes for cerebral transport and cellular uptake ofl-tryptophan, and because of substrate inhibition on tryptophan hydroxylase, excessively high doses of tryptophan may actually decrease the production of cerebral serotonin and 5-hydroxyindoleacetic acid.Some aspects of this paper were presented in a lecture at the meeting of the International Study Group for Tryptophan Research (ISTRY-77) on August 11, 1977 at the University of Wisconsin, Madison, Wisconsin.     

Evidence for presence of kynurenine in lung and brain of neonate hamsters

Using immunocytochemical techniques, we report here direct evidence of kynurenine (Kyn) presence and localization in the lung and brain. Kyn is a metabolite of tryptophan and 5-hydroxytryptophan, produced by indoleamine 2,3-dioxygenase (IDO). Whereas IDO has been quantitated in tissues from lung, brain, and other organs, Kyn has only been identified in brain (by HPLC), and its specific localization has not been determined. We reacted alternate serial paraffin sections with antisera raised in rabbits against a L-Kyn-albumin conjugate, and with anti-5HT (serotonin, 5-hydroxytryptamine), using the PAP method. Kyn-like immunoreactivity in the lung was specifically localized to cells of the bronchiolar epithelium resembling basal cells. Taller epithelial cells in the bronchi and dorsal trachea were likewise positive whereas neuroepithelial bodies were negative. Immunoreactivity in the brain was typically localized to cells localized in the ependyma of the walls of all ventricles, and to nerve fibers. The cellular Kyn-like reactivity was totally separate from that of anti-5HT, the latter uniquely staining argyrophil lung neuroendocrine cells and raphae neurons of the brain. Our findings suggest a route of tryptophan metabolism in the lung and brain alternate to the common pathway leading to 5-hydroxyindoleacetic acid via 5-HT. This route is of physiologic and pathologic significance as many metabolites are pharmacologically active.     

Binding of nitric oxide to reduced L-tryptophan-2,3-dioxygenase as studied by electron paramagnetic resonance.

Ferrous L-tryptophan-2,3-dioxygenase reacts with nitric oxide both in the presence and in the absence of L-tryptophan. Electron paramagnetic resonance studies suggest that the proximal ligand of the heme is a nitrogen atom, probably from an histidyl residue. The interaction of the protein with substrate changes both the symmetry of the paramagnetic center and the mode of interaction of the iron atom with its two axial ligands, NO and the proximal nitrogen atom. Optical absorption and EPR spectra suggest that the affinity of NO for tryptophan dioxygenase increases in the order: tryptophan dioxygenase, tryptophan dioxygenase + alpha-methyltryptophan, tryptophan diogenase " 5-hydroxytryptophan, tryptophan dioxygenase + L-tryptophan. A possible correlation between the number of superhyperfine lines in the EPR spectrum and the affinity of the enzyme for NO is discussed.     

The role of tryptophan 2,3-dioxygenase in the hormonal control of tryptophan metabolism in isolated rat liver cells. Effects of glucocorticoids and experimental diabetes.

The metabolism of L-tryptophan by isolated liver cells prepared from control, adrenalectomized, glucocorticoid-treated, acute-diabetic, chronic-diabetic and insulin-treated chronic-diabetic rats was studied. Liver cells from adrenalectomized rats metabolized tryptophan at rates comparable with the minimum diurnal rates of controls, but different from rates determined for cells from control rats 4h later. Administration of dexamethasone phosphate increased the activity of tryptophan 2,3-dioxygenase (EC 1.13.11.11) 7-8-fold, and the flux through the kynurenine pathway 3-4-fold, in cells from both control and adrenalectomized rats. Increases in flux through kynureninase (EC 3.7.1.3) and to acetyl-CoA can be explained in terms of increased substrate supply from tryptophan 2,3-dioxygenase. The metabolism of tryptophan was increased 3-fold in liver cells isolated from acutely (3 days) diabetic rats, with a 7-8-fold increase in the maximal activity of tryptophan 2,3-dioxygenase. The oxidation of tryptophan to CO2 and metabolites of the glutarate pathway increased 4-5-fold, consistent with an increase in picolinate carboxylase (EC 4.1.1.45) activity. Liver cells isolated from chronic (10 days) diabetic rats metabolized tryptophan at rates comparable with those of cells from acutely diabetic rats, but with a 50% decrease in the activity of tryptophan 2,3-dioxygenase. The proportion of flux from tryptophan 2,3-dioxygenase to acetyl-CoA, however, was increased by 50%; this was indicative of further increases in the activity of picolinate carboxylase. Administration of insulin partially reversed the effects of chronic diabetes on the activity of tryptophan 2,3-dioxygenase and flux through the kynurenine pathway, but had no effect on the increased activity of picolinate carboxylase. The role of tryptophan 2,3-dioxygenase in regulating the blood tryptophan concentration is discussed with reference to its sensitivity to the above conditions.     

Alterations of tryptophan metabolism in a rat strain (Osborne-Mendel) predisposed to obesity

1. Osborne-Mendel (O-M) rats displayed differences in brain and systemic tryptophan metabolism. O-M rats had decreased brainstem tryptophan-5-hydroxylase activity and decreased serotonin (5-HT) levels as compared to Sprague-Dawley rats. However, brain tryptophan levels were actually increased in O-M rats. Norepinephrine, dopamine and 5-hydroxyindole-3-acetic acid levels were not different between strains. 2. Pineal serotonin levels were increased in O-M rats. 3. Liver tryptophan 2,3-dioxygenase activity was increased in O-M rats while tyrosine aminotransferase activity was not different between strains. 4. Total blood cholesterol was decreased in O-M rats while triglycerides, free fatty acids and albumin was not different between strains. Total serum tryptophan was not different between strains while O-M rats had an increased level of free (unbound) tryptophan.     

Enzyme activities involved in tryptophan metabolism along the kynurenine pathway in rabbits

The following enzyme activities of the tryptophan-nicotinic acid pathway were studied in male New Zealand rabbits: liver tryptophan 2,3-dioxygenase, intestine indole 2,3-dioxygenase, liver and kidney kynurenine 3-monooxygenase, kynureninase, kynurenine-oxoglutarate transaminase, 3-hydroxyanthranilate 3,4-dioxygenase, and aminocarboxymuconate-semialdehyde decarboxylase. Intestine superoxide dismutase and serum tryptophan were also determined. Liver tryptophan 2,3-dioxygenase exists only as holoenzyme, but intestine indole 2,3-dioxygenase is very active and can be considered the key enzyme which determines how much tryptophan enters the kynurenine pathway also under physiological conditions. The elevated activity of indole 2,3-dioxygenase in the rabbit intestine could be related to the low activity of superoxide dismutase found in intestine. Kynurenine 3-monooxygenase appeared more active than kynurenine-oxoglutarate transaminase and kynureninase, suggesting that perhaps a major portion of kynurenine available from tryptophan may be metabolized to give 3-hydroxyanthranilic acid, the precursor of nicotinic acid. In fact, 3-hydroxyanthranilate 3,4-dioxygenase is much more active than the other previous enzymes of the kynurenine pathway. In the rabbit liver 3-hydroxyanthranilate 3,4-dioxygenase and aminocarboxymuconate-semialdehyde decarboxylase show similar activities, but in the kidney 3-hydroxyanthranilate 3,4-dioxygenase activity is almost double. These data suggest that in rabbit tryptophan is mainly metabolized along the kynurenine pathway. Therefore, the rabbit can also be a suitable model for studying tryptophan metabolism in pathological conditions.     

Indoleamine 2,3-dioxygenase. Formation of L-kynurenine from L-tryptophan in cultured rabbit fineal gland

The distribution of the indoleamine 2,3-dioxygenase activity was investigated in various parts of the rabbit brain using the supernatant fraction (30,000 X g, 30 min) of homogenates. A low but significant activity was detected in all parts of the brain. The highest activity was associated with the pineal gland and choroid plexus. Specific activities of the supernatant fractions derived from the pineal gland and choroid plexus were 84.8 and 34.2 pmol/h/mg of protein at 37 degrees C, respectively, with L-tryptophan as substrate. When the pineal gland was cultured with L-[methylene-14C]tryptophan, L-[methylene-14C]kynurenine formed by the action of indoleamine 2,3-dioxygenase was found as one of the major products. It was isolated by DEAE-cellulose column chromatography and identified by thin layer chromatography with and without the treatment by kynureninase from a pseudomonad. The amount of kynurenine thus measured accounted for approximately one-third of the total amount of tryptophan metabolites, indicating that the kynurenine pathway is one of the major metabolic pathways of tryptophan in the rabbit pineal gland.     

Interaction of tryptophan and related compounds with oxygen- and carbon-centered radicals

Abstract

Effects of tryptophan, 5-hydroxytryptophan, serotonin, and melatonin on final product formation during radiolysis of deaerated and oxygen-saturated ethanol and aqueous 1M ethanol solutions were studied. The named amino acids were found to be capable of adding α-hydroxyethyl radicals, thereby suppressing recombination reactions of these species. Unlike melatonin, tryptophan, 5-hydroxytryptophan, and serotonin were able to reduce oxygen-centered radicals being formed on radiation-chemical oxidation of ethanol in the presence of oxygen via electron transfer from the amine nitrogen lone pair.     

Effect of 8-hydroxy-2-(di-n-propylamino)-tetralin on the tryptophan-induced increase in 5-hydroxytryptophan accumulation in rat brain

The injection of 8-hydroxy-2-(di-n-propylamino)-tetralin [8-OH-DPAT]reduced 5-hydroxytryptophan accumulation in vivo in rat cerebral cortex, hypothalamus and brainstem. Brain tryptophan levels were unaffected. Dose-related increases in 5-hydroxytryptophan accumulation produced by single injections of L-tryptophan (0, 25, 75 mg/kg ip) were substantially diminished by pretreatment with 8-OH-DPAT. The drug did not affect the tryptophan-induced increments in brain tryptophan level. Since 8-OH-DPAT is known to reduce the activity of serotonin neurons in vivo, these results suggest that when serotonin neurons are relatively inactive, the ability of an injection of tryptophan to stimulate serotonin synthesis is greatly attenuated.     

Stimulation of the Serotonin Autoreceptor Prevents the Calcium-Calmodulin-Dependent Increase of Serotonin Biosynthesis in Rat Raphe Slices

The role of the serotonin (5-hydroxytryptamine) autoreceptor in the regulation of the activity of tryptophan hydroxylase was investigated in rat raphe slices. The activity of tryptophan hydroxylase was estimated by measuring the accumulation of 5-hydroxytryptophan in the presence of inhibition of aromatic L-amino acid decarboxylase using 3-hydroxy-4-bromobenzyloxy-amine by HPLC with fluorescence detection. Serotonin and its agonists N,N-dimethyl-5-methoxytryptamine and 1-(m-chlorophenyl)-piperazine reduced the formation of 5-hydroxytryptophan to 50-60% at 10(-5) M. The effect of serotonin was reversed by 10(-5) M methiothepin, an antagonist of the serotonin autoreceptor. The calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) and N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), dose-dependently reduced the basal formation of 5-hydroxytryptophan to 40-50% at 10(-6) and 10(-4) M, respectively. W-7 also reduced the activated formation by A-23187 or dibutyryl cyclic AMP in a dose-dependent manner. W-7 had no effect on 5-hydroxytryptophan formation reduced by serotonin at 10(-5) M. These results suggest that the role of the serotonin autoreceptor was related to the prevention of the calcium-calmodulin-dependent activation of tryptophan hydroxylase.     

Inhibition of indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase by β-carboline and indole derivatives

β-Carboline derivatives inhibited both indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase activities from various sources. Among them, norharman is most potent for both enzymes from mammalian sources. Kinetic studies revealed that norharman is uncompetitive (K_i = 0.12 mm) with l-tryptophan for rabbit intestinal indoleamine 2,3-dioxygenase, and linearly competitive (K_i = 0.29 mm) with l-tryptophan for mouse liver tryptophan 2,3-dioxygenase. In addition, some β-carbolines selectively inhibited one enzyme or the other. Pseudomonad tryptophan 2,3-dioxygenase was inhibited by a different spectrum of β-carbolines. Such a selective inhibition by the structure of substrate analogs is more evident by the use of indole derivatives. Indole-3-acetamide, indole-3-acetonitrile and indole-3-acrylic acid exhibited a potent inhibition for mammalian tryptophan 2,3-dioxygenase, while they moderately inhibited the pseudomonad enzyme. However, they showed no inhibition for indoleamine 2,3-dioxygenase. These results suggest the difference of the structures of the active sites among these enzymes from various sources.     

Characterization of TioF, a tryptophan 2,3-dioxygenase involved in 3-hydroxyquinaldic acid formation during thiocoraline biosynthesis

Thiocoraline is a thiodepsipeptide antitumor agent that belongs to the family of bisintercalator natural products that bind duplex DNA through their two planar intercalating moieties. In thiocoraline, the 3-hydroxyquinaldic acid (3HQA) chromophores required for intercalation are derived from L-Trp. We have expressed the Micromonospora sp. ML1 tryptophan 2,3-dioxygenase(TDO) TioF, purified it from E. coli, and confirmed its role in the irreversible oxidation of L-Trp to N-formylkynurenine, the proposed first step during 3HQA biosynthesis. We have established that TioF is a catalyst with broader specificity than other TDOs, but that is less promiscuous than indoleamine 2,3-dioxygenases. TioF was found to display activity with various L-Trp analogs (serotonin, D-Trp, and indole). The TioF reaction products generated during this study will be used as substrates for subsequent analysis of the other enzymes involved in 3HQA biosynthesis.     

Brain serotonin and blood pressure regulation: studies using in vivo electrochemistry and direct tissue assay

Hypotensive responses to tryptophan and 5-hydroxytryptophan infusions were studied in normotensive male Sprague-Dawley rats. Results showed that 5-hydroxytryptophan but not tryptophan lowered pressure in a dose dependent way in direct relation to the production of brain serotonin and 5-HIAA. Intrinsic release of serotonin from brain was also studied during periods of induced hypertension and hypotension. Brain monoamine responses to blood pressure changes induced by intravenous phenylephrine and nitroprusside were measured in dorsal raphe nucleus and nucleus tractus solitarius by in vivo electrochemistry. Results showed that 5-HIAA was increased during drug induced hypertension and during reflex hypertension which followed a period of hypotension. These changes were blocked by sinoaortic denervation indicating that these central serotonergic neurons are responding to increased pressure sensed by baroreceptors. Therefore, serotonin has a role in blood pressure regulation as a pharmacologic agent and as a neurotransmitter in homeostatic control of pressure.     

The metabolism of L-tryptophan by isolated rat liver cells. Quantification of the relative importance of, and the effect of nutritional status on, the individual pathways of tryptophan metabolism.

1. The metabolism of L-tryptophan by liver cells prepared from fed and 48 h-starved rats was studied. Methods are described, with the use of L-[ring-2-(14)C], L-[carboxy-14C]-and L-[benzene-ring-U-14C]-tryptophan, for the simultaneous determination of tryptophan 2,3-dioxygenase and kynureninase activities and of the oxidation of tryptophan to CO2 and non-aromatic intermediates of the kynurenine-glutarate pathway. 2. At physiological concentrations (0.1 mM), tryptophan was oxidized by tryptophan 2,3-dioxygenase at comparable rates in liver cells from both fed and starved rats. Kynureninase activity of hepatocytes from starved rats was 50% greater than that of cells from fed rats. About 10% of the tryptophan metabolized by tryptophan 2,3-dioxygenase was degraded completely to CO2. 3. In the presence of 0.5 mM-L-tryptophan, tryptophan 2,3-dioxygenase and kynureninase activities increased 5--6-fold. Liver cells from starved rats oxidized tryptophan at about twice the rate of these from fed rats. Degradation of tryptophan to non-aromatic intermediates of the glutarate pathway and CO2 was increased only 3-fold, suggesting an accumulation of aromatic intermediates of the kynurenine pathway. 4. Rates of metabolism with 2.5 mM-L-tryptophan were not significantly different from those obtained with 0.5 mM-tryptophan. 5. Rates of synthesis of quinolinic acid from 0.5 mM-L-tryptophan, determined either by direct quantification or indirectly from rates of radioisotope release from L-[carboxy-(14)C]- and [benzene-ring-U-14C]tryptophan, were essentially similar. 6. At all three concentrations examined, tryptophan was degraded exclusively through kynurenine; there was no evidence of formation of either indol-3-ylacetic acid or 5-hydroxyindol-3-ylacetic acid.     

Determination of 5-hydroxytryptophan, 5-hydroxytryptamine, and 5-hydroxyindoleacetic acid in 20 rat brain nuclei using liquid chromatography with electrochemical detection

High-performance liquid chromatography with electrochemical detection is utilized for the simultaneous determination of serotonin, its precursor 5-hydroxytryptophan, and its major metabolite 5-hydroxyindoleacetic acid in nervous tissue samples. Tissue preparation required only homogenization in acidic solution and centrifugation prior to application to the chromatograph. Detection limits in the low picogram range were obtained for those indoles separated. This assay was used in combination with a micropunch dissection technique of 20 discrete rat brain nuclei to measure serotonin, its precursor, and major metabolite. The specificity of the assay was checked with pharmacological experiments aimed to increase or decrease serotonin levels. Pargyline, a monoamine oxidase inhibitor, led to a marked increase in serotonin and a decrease of 5-hydroxyindoleacetic acid while p-chlorophenylalanine, by blocking the conversion of tryptophan to 5-hydroxytryptophan, selectively depleted 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid.     

Acetaminophen inhibits liver trytophan-2,3-dioxygenase activity with a concomitant rise in brain serotonin levels and a reduction in urinary 5-hydroxyindole acetic acid

The effect of the analgesic agent, acetaminophen was determined on rat forebrain serotonin levels as well as hepatic tryptophan-2,3-dioxygenase (TDO) activity and urinary 5-hydroxyindole acetic acid (5-HIAA). The results show that acetaminophen administration (100mg/kg) over three hours does not affect the holoenzyme of tryptophan-2,3-dioxygenase but significantly inhibits the apoenzyme. This inhibition is accompanied by a concomitant rise in forebrain serotonin levels. This phenomenon is also accompanied by a reduction in urinary 5-HIAA levels. These results suggest that acetaminophen use is accompanied by changes in brain serotonin levels due to inhibition of hepatic tryptophan-2,3-dioxygenase activity. This in turn could explain the possible abuse potential of acetaminophen and its effects on mood at high doses.     

One-step production of picolinic acids from 2-aminophenols catalyzed by 2-aminophenol 1,6-dioxygenase

Picolinic acids have been synthesized previously from catechols by the action of catechol 2,3-dioxygenase and a subsequent chemical reaction in the presence of ammonia. 2-Aminophenol 1,6-dioxygenase catalyzes ring cleavage of several ortho-aminophenols. The ring fission products spontaneously convert to picolinic acids. Resting cells of Escherichia coli DH5α/pNBZ14 harboring the genes for 2-aminophenol 1,6-dioxygenase converted 2-aminophenol and 6-amino-m-cresol to picolinic acid and 5-methylpicolinic acid with yields greater than 90%. The results provide a convenient strategy for the synthesis of substituted picolinic acids from the corresponding aminophenols. Journal of Industrial Microbiology & Biotechnology (2000) 25, 25–28. Received 25 October 1999/ Accepted in revised form 19 April 2000     

Serotonin synthesis and its light-dark variation in the rat retina

Retinal circadian rhythms are driven by an intrinsic oscillator, using chemical signals such as melatonin, secreted by photoreceptor cells. The purpose of the present work was to identify the origin of serotonin, the precursor of melatonin, in the retina of adult rat, where no immunoreactivity for serotonin or tryptophan hydroxylase had ever been detected. To demonstrate local synthesis of serotonin in the rat retina, substrates of tryptophan hydroxylase, the first limiting enzyme in the serotonin pathway, have been used. Tryptophan, in the presence of an inhibitor of aromatic amino acid decarboxylase, enhanced 5-hydroxytryptophan levels, whereas alpha-methyltryptophan, a competitive substrate inhibitor, was hydroxylated into alpha-methyl-5-hydroxytryptophan. Tryptophan hydroxylase substrate concentration was higher in the dark period than in the light period, and formation of hydroxylated compounds was increased. The presence of tryptophan hydroxylase mRNA in the rat retina was confirmed by RT-PCR. Taken together, the results support the local synthesis of serotonin by tryptophan hydroxylation, this metabolic pathway being required more critically when 5-HT is used for melatonin synthesis.