Kynureninase-Type enzymes of Penicillum roqueforti, Aspergillus niger, Rhizopus stolonifer, and Pseudomonas fluorescens: further evidence for distinct kynureninase and hydroxykynureninase activities.

The kynureninase-type enzymes of three fungi and one bacterium were isolated and examined kinetically for their ability to catalyze the hydrolysis of L-kynurenine and L-3-hydroxykynurenine. The phycomycete Rhizopus stolonifer was found to contain a single, constitutive enzyme with Km for L-3-hydroxykynurenine and L-kynurenine of 6.67 times 10-minus 6 and 2.5 times 10-minus 4 M, respectively. The ascomycetes Aspergillus niger and Penicillium roqueforti each contain an enzyme, induced by L-tryptophan, with similar Km for L-3-hydroxykynurenine and L-kynurenine ranging from 5.9 times 10-minus 5 to 14.3 times 10-minus 5 M, as well as a constitutive enzyme with Km for the two substrates of similar to 4 times 10-minus 6 M and 10-minus 4 M. The bacterium Pseudomonas fluorescens has a single, inducible enzyme with Km for L-3-hydroxykynurenine and L-kynurenine of 5 times 10-minus 4 and 7 times 10-minus 5 M. In addition, significant differences in maximal velocities (Vmax) were observed in two cases. The Vmax of the inducible activity from P. fluorescens was 4.5 times greater for L-kynurenine than L-3-hydroxykynurenine, whereas the Vmax of the constitutive activity from R. stolonifer was 2.5 times greater for L-3-hydroxykynurenine. It is concluded (i) that the constitutive activities are hydroxykynureninases involved in the biosynthesis of nicotinamide adenine dinucleotide from L-tryptophan, (ii) that the inducible activities are kynureninases involved in the catabolism of L-tryptophan to anthranilate, and (iii) that R. stolonifer and P. fluorescens, respectively, carry the most specific examples of each type of enzyme.     

Relationship between L-tryptophan uptake and L-tryptophan 2,3-dioxygenase activity in rat hepatocytes

The relationship between L-tryptophan uptake and tryptophan 2,3-dioxygenase activity in hepatocytes was examined and compared with the change of hepatic L-leucine, L-phenylalanine, and L-tyrosine uptakes using isolated hepatocytes of rats in which the oxygenase was induced with L-tryptophan or hydrocortisone. In L-tryptophan- or hydrocortisone-treated rat hepatocytes, the rate of L-tryptophan uptake into hepatocytes via the saturable high-affinity transport component significantly increased but the hepatic uptake rate of L-leucine did not change at all. In hydrocortisone-treated rat hepatocytes, a little stimulated hepatic uptake of L-phenylalanine or L-tyrosine was observed. In the stimulated hepatic uptake of L-tryptophan via the high-affinity transport component, the Km value did not change but the Vmax value increased. Liver plasma membranes prepared from rats treated with L-tryptophan or hydrocortisone showed the same binding rate of L-tryptophan to the membranes as those from control rats. In addition, hepatic L-tryptophan uptake via the high-affinity transport component correlated well with hepatic tryptophan 2,3-dioxygenase activity (r = 0.787). The present results indicate that the uptake of L-tryptophan into hepatocytes via a transport system which works under physiological conditions is closely related to hepatic tryptophan 2,3-dioxygenase activity.     

Effects of systemic and central nervous system localized inflammation on the contributions of metabolic precursors to the L-kynurenine and quinolinic acid pools in brain

L-Kynurenine and quinolinic acid are neuroactive L-tryptophan-kynurenine pathway metabolites of potential importance in pathogenesis and treatment of neurologic disease. To identify precursors of these metabolites in brain, [(2)H(3) ]-L-kynurenine was infused subcutaneously by osmotic pump into three groups of gerbils: controls, CNS-localized immune-activated, and systemically immune-activated. The specific activity of L-kynurenine and quinolinate in blood, brain and systemic tissues at equilibrium was then quantified by mass spectrometry and the results applied to a model of metabolism to differentiate the relative contributions of various metabolic precursors. In control gerbils, 22% of L-kynurenine in brain was derived via local synthesis from L-tryptophan/formylkynurenine versus 78% from L-kynurenine from blood. Quinolinate in brain was derived from several sources, including: local tissue L-tryptophan/formylkynurenine (10%), blood L-kynurenine (35%), blood 3-hydroxykynurenine/3-hydroxyanthranilate (7%), and blood quinolinate (48%). After systemic immune-activation, however, L-kynurenine in brain was derived exclusively from blood, whereas quinolinate in brain was derived from three sources: blood L-kynurenine (52%), blood 3-hydroxykynurenine or 3-hydroxyanthranilate (8%), and blood quinolinate (40%). During CNS-localized immune activation, > 98% of both L-kynurenine and quinolinate were derived via local synthesis in brain. Thus, immune activation and its site determine the sources from which L-kynurenine and quinolinate are synthesized in brain. Successful therapeutic modulation of their concentrations must take into account the metabolic and compartment sources.     

Purification and characterization of kynurenine--2-oxoglutarate aminotransferase from the liver, brain and small intestine of rats.

1. Kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) was purified to homogeneity from the liver, brain and small intestine of rats by the same procedure. The three enzyme preparations had nearly identical pH optima, substrate specificities and molecular weights. Isoenzyme 1 was active with 2-oxoglutarate but not with pyruvate as amino acceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order to activity: L-aspartate greater than L-tyrosine greater than L-phenylalanine greater than L-tryptophan greater than 5-hydroxy-L-tryptophan greater than L-kynurenine. The molecular weight was approximately 88 000 as determined by sucrose-density-gradient centrifugation. The pH optimum was between 8.0 and 8.5. On the basis of substrate specificity, substrate inhibition, subcellular distribution and polyacrylamide-disc-gel electrophoresis, it is suggested that liver, brain and small intestinal kynurenine-2-oxoglutarate aminotransferase (isoenzyme 1) is identical with mitochondrial tyrosine-2-oxoglutarate aminotransferase and also with mitochondrial aspartate-2-oxoglutarate aminotransferase. 2. An additional kynurenine-2-oxoglutarate aminotransferase (isoenzyme 2) was purified from the liver. This enzyme was specific for 2-oxoglutarate and L-kynurenine. Sucrose-density-gradient centrifugation gave a molecular weight of approximately 100 000. The pH optimum was between 6.0 and 6.5. This enzyme was not detected in the brain or small intestine.     

Peripheral distribution of kynurenine metabolites and activity of kynurenine pathway enzymes in renal failure.

We investigated L-kynurenine distribution and metabolism in rats with experimental chronic renal failure of various severity, induced by unilateral nephrectomy and partial removal of contralateral kidney cortex. In animals with renal insufficiency the plasma concentration and the content of L-tryptophan in homogenates of kidney, liver, lung, intestine and spleen were significantly decreased. These changes were accompanied by increase activity of liver tryptophan 2,3-dioxygenase, the rate-limiting enzyme of kynurenine pathway in rats, while indoleamine 2,3-dioxygenase activity was unchanged. Conversely, the plasma concentration and tissue content of L-kynurenine, 3-hydroxykynurenine, and anthranilic, kynurenic, xanthurenic and quinolinic acids in the kidney, liver, lung, intestine, spleen and muscles were increased. The accumulation of L-kynurenine and the products of its degradation was proportional to the severity of renal failure and correlated with the concentration of renal insufficiency marker, creatinine. Kynurenine aminotransferase, kynureninase and 3-hydroxyanthranilate-3,4-dioxygenase activity was diminished or unchanged, while the activity of kynurenine 3-hydroxylase was significantly increased. We conclude that chronic renal failure is associated with the accumulation of L-kynurenine metabolites, which may be involved in the pathogenesis of certain uremic syndromes.     

Catabolism of tryptophan, anthranilate, and 2,3-dihydroxybenzoate in Trichosporon cutaneum.

Trichosporon cutaneum degraded L-tryptophan by a reaction sequence that included L-kynurenine, anthranilate, 2,3-dihydroxybenzoate, catechol, and beta-ketoadipate as catabolites. All of the enzymes of the sequence were induced by both L-tryptophan and salicylate, and those for oxidizing kynurenine and its catabolites were induced by anthranilate but not by benzoate; induction was not coordinate. Molecular weights of 66,100 and 36,500 were determined, respectively, for purified 2,3-dihydroxybenzoate decarboxylase and its single subunit. Substrates for this enzyme were restricted to benzoic acids substituted with hydroxyl groups at C-2 and C-3; no added coenzyme was required for activity. Partially purified anthranilate hydroxylase (deaminating) catalyzed the incorporation of one atom of 18O, derived from either 18O2 or H2(18)O, into 2,3-dihydroxybenzoic acid.     

Indoleamine 2,3-dioxygenase activity and L-tryptophan transport in human breast cancer cells

The activity and expression of indoleamine 2,3-dioxygenase together with L-tryptophan transport has been examined in cultured human breast cancer cells. MDA-MB-231 but not MCF-7 cells expressed mRNA for indoleamine 2,3-dioxygenase. Kynurenine production by MDA-MB-231 cells, which was taken as a measure of enzyme activity, was markedly stimulated by interferon-gamma (1000 units/ml). Accordingly, L-tryptophan utilization by MDA-MB-231 cells was enhanced by interferon-gamma. 1-Methyl-DL-tryptophan (1 mM) inhibited interferon-gamma induced kynurenine production by MBA-MB-231 cells. Kynurenine production by MCF-7 cells remained at basal levels when cultured in the presence of interferon-gamma. L-Tryptophan transport into MDA-MB-231 cells was via a Na(+)-independent, BCH-sensitive pathway. It appears that system L (LAT1/CD98) may be the only pathway for l-tryptophan transport into these cells. 1-Methyl-D,L-tryptophan trans-stimulated l-tryptophan efflux from MDA-MB-231 cells and thus appears to be a transported substrate of system L. The results suggest that system L plays an important role in providing indoleamine-2,3-dioxygenase with its main substrate, L-tryptophan, and suggest a mechanism by which estrogen receptor-negative breast cancer cells may evade the attention of the immune system.     

Purification and characterization of yeast L-kynurenine aminotransferase with broad substrate specificity

L-Kynurenine aminotransferase [L-kynurenine:2-oxoglutarate aminotransferase (cyclizing), EC 2.6.1.7] has been purified to homogeneity and crystallized from cell-free extracts of a yeast, Hansenula schneggii, grown in a medium containing L-tryptophan as an inducer. The enzyme has a molecular weight of about 100,000 and consists of two subunits identical in molecular weight (52,000). The enzyme exhibits absorption maxima at 280, 335, and 430 nm, and contains 2 mol of pyridoxal 5'-phosphate per mol of enzyme. The enzyme-bound pyridoxal 5'-phosphate shows negative circular dichroic extrema, in contrast with other pyridoxal 5'-phosphate acting on L-amino acids. In addition to L-kynurenine and alpha-ketoglutarate, which are the most preferred substrates, a large number of L-amino acids and alpha-keto acids can serve as substrates; the extremely broad substrate specificity is the most characteristic feature of this yeast enzyme. The enzyme activity is significantly affected by both carbonyl and sulfhydryl reagents. Certain dicarboxylic acids such as adipate and pimelate act as competitive inhibitors. Addition of various substrate amino acids to the culture medium results in the inductive formation of aminotransferases which are immunochemically indistinguishable from L-kynurenine aminotransferase.     

Kynurenine pyruvate transaminase and its inhibitor in rat intestine

Kynurenine pyruvate transaminase was found to be present in rat small intestine, partially purified and characterized. The enzyme catalysed the conversion of L-kynurenine to kynurenic acid. Transamination rates of 3-hydroxy-DL-kynurenine and 5-hydroxy-DL-kynurenine by the enzyme were 1/2.9 and 1/2.6 that of L-kynurenine. The enzyme showed higher preference for pyruvate than 2-oxoglutarate as aminoacceptor. The pH optimum of the reaction was 8.0 to 8.5. Purification of the enzyme lowered markedly apparent Km for L-kynurenine but not for pyruvate. It was shown that the inhibitor of kynurenine pyruvate transaminase was present in the intestine, on the basis of the inhibition produced by heating a portion of each purification step enzyme preparation in 50% ethanol, centrifuging, concentrating it, and adding it to an incubate of the unheated preparation. The possible interrelationship of enzyme and inhibitor was discussed and comparisons with kynurenine transaminase in liver, kidney and brains were noted.     

Studies on the kynurenine adminotransferase activity in rat liver and kidney.

The kynurenine aminotransferase activity of supernatant and mitochondrial fractions obtained from rat liver and kidney was studied with L-kynurenine and L-3-hydroxykynurenine as substrates. A substrate inhibition with L-kynurenine at concentrations higher than 6-7mM was observed with all four enzyme preparations. This did not happen with L-3-hydroxykynurenine as a substrate. Moreover, the liver mitochondrial enzyme shows a Km for pyridoxal phosphate 2-4 times smaller than the other preparations when assayed with L-3-hydroxykynurenine as a substrate. Therefore, the accumulation of xanthurenic acid and not of kynurenic acid in B6 deficiency could be related both to this high activity of liver mitochondrial kynurenine aminotransferase with L-3-hydroxykynurenine, even at small concentrations of B6, and to substrate inhibition observed with L-kynurenine and not with L-3-hydroxykynurenine.     

Indoleamine 2,3-diooxygenase in periaortic fat: mechanisms of inhibition of contraction

Indoleamine 2,3-dioxygenase (IDO) metabolizes L-tryptophan to L-kynurenine, promotes immunosuppression, and has been described as a consumer of superoxide. We discovered IDO expression in periaortic fat and tested the hypothesis that periarterial IDO functionally reduces agonist-induced contraction. Our model was the thoracic aorta, abdominal aorta, and superior mesenteric artery of the male Sprague-Dawley rat. Periaortic fat from the thoracic aorta stained intensely for IDO, the brown fat marker uncoupling protein-1, and oil red O as a general lipid marker. White fat around the mesenteric artery and abdominal aorta stained less for IDO; brown fat was less abundant. IDO activity (kynurenine-to-tryptophan ratio via HPLC) was detected in visceral and mesenteric artery fat (ratio: ～4) but was highest in perithoracic aortic fat (ratio: 10 ± 1.1). In isometric contractile experiments, periadventitial fat reduced ANG II-induced thoracic aortic (with fat: 34% of without fat) and mesenteric artery (with fat: 63% of without fat) maximal contraction. In contrast, periadventitial fat did not reduce agonist-induced contraction in the abdominal aorta. The IDO inhibitor 1-L-methyltryptophan (1-MT) reversed the fat-induced reduction of ANG II-induced contraction in the thoracic aorta but not in the mesenteric artery. The IDO metabolite kynurenine relaxed the thoracic aorta only at high (9 mM) concentrations, whereas the downstream metabolite quinolinic acid (1 mM) relaxed the contracted thoracic aorta (～80%). 1-MT did not correct the reduction in basal superoxide levels observed in the presence of perithoracic aortic fat. We conclude that IDO is an enzyme active primarily in brown fat surrounding the thoracic aorta and depresses aortic contractility.     

Glucosinolate Biosynthesis (Further Characterization of the Aldoxime-Forming Microsomal Monooxygenases in Oilseed Rape Leaves)

The initial steps in glucosinolate biosynthesis are thought to proceed from amino acids, via N-hydroxy amino acids, to aldoximes. We showed previously that microsomes from green leaves of oilseed rape (Brassica napus cv Bienvenu) contain two distinct monooxygenases that catalyze the conversion of homophenylalanine and dihomomethionine to their respective aldoximes. Further characterization of these enzymes has now demonstrated that the latter enzyme catalyzes the NADPH-dependent oxidative decarboxylation of two higher homologs of methionine, in addition to dihomomethionine. No activity was found for either enzyme with L-methionine, DL-homomethionine, L-phenylalanine, L-tyrosine, or L-tryptophan. Both of these rape monooxygenase activities are dependent on O2, not requiring any other O2 species or radical. The presence of an unoxidized sulfur atom and its relative position in the side chain of the aliphatic substrates are important for binding to the active site of the methionine-homolog enzyme. Neither enzyme has any characteristics of a cytochrome P450-type enzyme, and antiserum raised against cytochrome P450 reductase did not significantly inhibit monooxygenase activity.     

Characteristics of interferon induced tryptophan metabolism in human cells in vitro

Interferon-gamma-induced tryptophan metabolism of human macrophages was compared to ten human neoplastic cell lines of various tissue origin and to normal dermal human fibroblasts. Tryptophan and metabolites were determined in supernatants of cultures, after incubation for 48 h, by high-performance liquid chromatography with ultraviolet and fluorescence detection. With the exception of two cell lines (Hep G 2, hepatoma and CaCo 2, colon adenocarcinoma) in all of the ten other cells and cell lines tryptophan degradation was induced by interferon-gamma. Five of these ten formed only kynurenine (SK-N-SH, neuroblastoma; T 24, J 82, bladder carcinoma; A 431, epidermoid carcinoma; normal dermal fibroblasts), three formed kynurenine and anthranilic acid (U 138 MG, glioblastoma; SK-HEP-1, hepatoma; A 549, lung carcinoma). Only one line, A 498 (kidney carcinoma) showed the same pattern of metabolites as macrophages (kynurenine, anthranilic acid and 3-hydroxyanthranilic acid). Interferon-gamma regulated only the activity of indoleamine 2,3-dioxygenase. All other enzyme activities detected were independent of interferon-gamma, as shown by the capacity of the cells to metabolize L-kynurenine or N-formyl-L-kynurenine. Increasing the extracellular L-tryptophan concentration resulted in a marked induction of tryptophan degradation by macrophages. Contrarily, a significant decrease of the tryptophan degrading activity was observed when the extracellular L-tryptophan concentration was increased 2-fold with SK-N-SH, T 24 and J 82, 4-fold with A 431 and A 549 and 10-fold with U 138 MG and SK-HEP-1. The activity was unaffected by extracellular L-tryptophan with dermal fibroblasts and A 498. Though interferon-gamma was the most potent inducer of tryptophan metabolism, interferon-alpha and/or -beta showed small but distinct action on some of the cells. In all cells which reacted to interferon-gamma by enhanced expression of class I and/or class II major histocompatibility complex antigens tryptophan degradation was also inducible. These results demonstrate that induction of indoleamine 2,3-dioxygenase is a common feature of interferon-gamma action, that the extent of this induction is influenced by extracellular L-tryptophan concentrations and that indoleamine 2,3-dioxygenase is the only enzyme in the formation of 3-hydroxyanthranilic acid from tryptophan which is regulated by interferon-gamma.     

Modulation of Tryptophan Hydroxylase Activity by Phospholipids: Stimulation Followed by Inactivation

The activity of tryptophan hydroxylase from the rat brainstem was stimulated rapidly three- to fourfold by the addition of phosphatidylinositol or phosphatidylserine. However, the activity of the enzyme once stimulated was decreased gradually by subsequent incubation with the phospholipid at 37 degrees C, reaching a level below the original activity after 1 h of incubation. The presence of ferrous ion almost perfectly protected the enzyme against this phospholipid inactivation. The activity of the enzyme inactivated by incubation with the phospholipid was not only restored, but also increased further by incubation at 37 degrees C with ferrous ion and dithiothreitol. Gel filtration analysis revealed that the enzyme stimulated by phosphatidylinositol was eluted in a void volume together with the phospholipid vesicles, but the enzyme inactivated by incubation with phosphatidylinositol was eluted at a later region apart from the vesicles. These results, taken together, suggest the possible involvement of cellular membranes in the regulation of tryptophan hydroxylase in the central nervous system.     

Presence of a Pathway for the Biosynthesis of Auxin via Indole-3-Acetamide in Trifoliata Orange

The auxin-biosynthetic pathway from L-tryptophan to indole-3-aceticadd via indole-3-acetamide (IAM), found in plant-pathogenicbacteria such as Agrobacterium tumefaciens and Pseudomonas savastanoi,has not been found in plants. We attempted to detect the enzymaticactivities for this pathway in cell-free systems from varioustissues of trifoliata orange (Poncirus trifoliata Rafin.). Ahigh level of activity of LAM hydrolase, which catalyzes theconversion of IAM to indole-3-acetic acid, was observed in acrude extract prepared from young fruits one week after fullbloom. Using -naphthaleneacetamide as a competitor of IAM hydrolase,a simple assay system was developed for the detection of theconversion of L-tryptophan to IAM (tryptophan monooxygenaseactivity). When this system was used to assay cell-free extractsof young fruit of P. trifoliata, the conversion of L-tryptophanto IAM was clearly demonstrated by the presence of IAM amongreaction products, as demonstrated by GC/MS analysis and theincorporation of ¹⁴C-labeled L-tryptophan into an IAM fraction.This is the first report indicating the presence of an auxin-biosyntheticpathway via IAM in P. trifoliata. Furthermore, it is shown thatboth enzyme activities in auxin biosynthesis increased transientlyduring fruit development. (Received October 9, 1992; Accepted November 2, 1992)     

Negligible amount of copper in hepatic L-tryptophan 2,3-dioxygenase.

During the purification of L-tryptophan 2,3-dioxygenase, a protohemoprotein from rat liver, both copper and heme contents of the preparations were found to be progressively increased as purification proceeded. However, the greater part of copper was removed in the late stages of the purification giving a copper to heme ratio less than 0.4. The small amounts of copper could further be reduced by one-half, by a mild treatment of enzyme with chelators such as ethylenedi aminetetraacetate, without any accompanying decrease in enzymatic activity. Since the turnover number of these enzyme preparations expressed per mol of enzyme-bound heme, 200 to 277 min-1 at 25 degrees, were either comparable to or slightly higher than those reported with homogeneous enzyme preparations, the heme in the preparation was considered to be of fully active L-tryptophan 2,3-dioxygenase and, therefore, such a small ratio of copper to heme, 0.1 to 0.3, indicated that copper is not a constituent of L-tryptophan 2,3-dioxygenase of rat liver. The findings were thus inconsistent with the results of Brady et al. (Brady, F. O., Monaco, M. E. Forman, H. J. Schutz, G., and Feigelson, P. (1972) J. Biol. Chem. 247, 7915-7922), who found that L-tryptophan 2,3-dioxygenase contained 2 g atoms of copper and 2 mol of heme/mol of enzyme. Possible reasons for this discrepancy have been discussed.     

Demonstration of 5'-methylthioadenosine phosphorylase activity in various rat tissues. Some properties of the enzyme from rat lung.

An enzyme (5'-methylthioadenosine phosphorylase) that catalyzes the phosphorolytic cleavage of 5'-methylthioadenosine to 5-methylthioadenosine to 5-methylthioribose-1-phosphate and adenine was found in various rat tissues. Liver and lung had the highest enzyme activities and heart the lowest, most of the activity (greater than 90%) was recovered in soluble tissue fractions. The enzyme from rat lung was purified about 30-fold by pH treatment (NH4)2SO4 fractionation, and gel filtration. The enzyme did not require an added metal-ion for activity, and was not inhibited by EDTA. Many compounds were tested for their inhibitory effects; of these, ribose 1-phosphate, 2-deoxyribose 1-phosphate, fructose 1-phosphate, adenine and guanine were shown to inhibit. Kinetic patterns on reciprocal plots were linear as a function of the concentration of either 5'-methylthioadenosine or phosphate. More detailed kinetic studies suggested that the rat lung 5'-methylioadenosine phosphorylase catalyzes an equilibrium-ordered reaction, and that 5'-methylthioadenosine is the first substrate to bind and 5-methylthioribose-1-phosphate is the first product to be released.     

Effect of substrate and small doses of cortisone on the induction of Tryptophan 2,3-dioxygenase (author's transl)]

A number of enzymes are induced by steroid hormones. In this paper the reaction of tryptophan 2,3-dioxygenase is further analyzed. In particular we show in which way the substrate and low doses of cortisone cause an induction. 1) For the induction of tryptophan 2,3-dioxygenase in adrenalectomized rats by 2.5 mg cortisone/kg, the presence of the substrate is necessary as well. Under these conditions an induction of the enzyme can already be registered in the presence of 12.5 mg L-tryptophan/kg. 2) In animals treated before with cortisone, the enzyme maximum appears 30 min after L-tryptophan injection, The enhancement of enzyme activity in animals which are treated with 2.5 mg cortisone/kg before is blocked by actidione only until 30 min after L-tryptophan injection. 3) Experiments with antibodies in animals treated with a low dosis of cortisone show that L-tryptophan acts mainly via enzyme degradation or the saturation with the coenzyme hematin, respectively.