Stereospecificity of hepatic L-tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase [L-tryptophan--oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.     

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.     

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.     

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.     

Infrared spectra of carbon monoxide complexes of indoleamine 2,3-dioxygenase and L-tryptophan 2,3-dioxygenases. Effects of substrates on the CO-stretching frequencies

Carbonmonoxy indoleamine 2,3-dioxygenase from rabbit small intestine exhibited two CO stretch bands at 1953 and 1933 cm-1 with half-band widths (delta v 1/2) of both approximately 15 cm-1. Upon addition of an excess amount of L-tryptophan, the substrate, the spectrum changed into that with an intense single band at 1902 cm-1 with the delta v 1/2 of 15 cm-1. Carbonmonoxy L-tryptophan 2,3-dioxygenase of Pseudomonas acidovorans in the absence of L-tryptophan showed a fused CO stretch band which consists of two components at 1965 and 1958 cm-1 (delta v 1/2 for the fused band; 25 cm-1), which was converted into a sharp single band at 1968 cm-1 (delta v 1/2; 10 cm-1) upon addition of excess L-tryptophan. On the other hand, CO complex of rat liver L-tryptophan 2,3-dioxygenase in the absence of L-tryptophan gave a spectrum with a poorly defined peak around 1961 cm-1. By the addition of L-tryptophan, the spectrum changed into that with two distinct bands at 1972 and 1920 cm-1 (delta v 1/2; 6 and 13 cm-1, respectively). These spectra were insensitive to pH in a range where the enzymes were not denatured (neutral to near pH 9). The infrared spectra of the carbonmonoxy enzymes were also affected by the addition of certain effectors such as skatole and alpha-methyl-DL-tryptophan, which facilitate the binding of L-tryptophan to the catalytic site of intestinal and Pseudomonas enzymes, respectively. However, the changes were of different types from those by the saturating amount of L-tryptophan. Possible mechanisms for these phenomena are discussed in relation to the structure of the heme-CO complex in these heme-containing dioxygenases.     

A specific interaction of L-tryptophan with CO of CO-bound indoleamine 2,3-dioxygenase identified by resonance Raman spectroscopy

Indoleamine 2,3-dioxygenase (IDO) is a heme enzyme which catalyzes dioxygenation of l-Trp (tryptophan), yielding N-formylkynurenine. IDO thus plays a key role in l-Trp catabolism in mammals. In the present study, resonance Raman (RR) spectra of the reduced carbon monoxide- (CO-) bound form of IDO were measured in order to gain insights into the active site environment of O(2). Binding of CO to l-Trp-bound IDO causes a significant change in the electronic and RR spectra of the heme, indicating that the π* orbitals of the carbon atom of CO interact with π orbitals of Fe and the porphyrin. On the other hand, binding of CO to d-Trp-bound IDO does not induce the same change. This is also the case with substrate-free IDO. Based on the distinct absorption spectra and RR bands of the vibrational signature of CO (ν(CO), δ(FeCO), and ν(Fe-CO)) of the l-Trp-bound species relative to the other two species, it is confirmed that sterically constrained geometry of the Fe-O-O unit exists as previously reported (Terentis, A. C., et al. (2002) J. Biol. Chem. 277, 15788-15794). In contrast, binding of d-Trp does not induce such constraint. The comparable values of V(max) reported for l-Trp and d-Trp are interpreted as a result of a change in the rate-limiting step in the reaction cycle of the enzyme induced by the d-enantiomer relative to the l-enantiomer. Enhancements of the overtone and the combination Raman modes of the Fe-CO stretching vibration are evident. The anharmonicity of the Fe-CO stretching oscillator is significantly higher than those of oxygen carrier proteins. This is a specific character of IDO and might be responsible for the unique reactivity of this enzyme.     

Radiocopper in L-tryptophan 2,3-dioxygenase isolated from Pseudomonas acidovorans grown in the presence of 64Cu (II).

L-Tryptophan 2,3-dioxygenase (EC 1.13.11.11), isolated from L-tryptophan-induced Pseudomonas acidovorans, ATCC 11299b, which has been grown in a medium containing 64Cu(NO3)2, has been shown to contain radiocopper. At several stages of purification of the enzyme samples were taken, and these were subjected to disc acrylamide gel electrophoresis in the presence of 10 mM L-tryptophan. After electrophoresis the position of the yellow heme band, corresponding to tryptophan oxygenase, was visually located, and the gels were sliced and counted. A large peak of radioactivity was seen to occur at the location on the gel of tryptophan oxygenase no matter what the stage of purification. Treatment of each sample before electrophoresis for 30 min at 37 degrees with gamma-globulins prepared from rabbits sensitized to homogeneous pseudomonad tryptophan oxygenase greatly reduced this peak of radioactivity, whereas treatment of each sample with rabbit preimmune gamma-globulin did not. This direct demonstration of the presence of coper in pseudomonad tryptophan oxygenase, using 64-Cu, avoided the problems and artifacts inherent in the usual techniques of copper analysis and unequivocally refutes the recent contention of Ishimura and Hayaishi ((1973) J. Biol.Chem. 248, 8610-8612) "that copper is not an essential component of L-tryptophan 2,3-dioxygenase of Pseudomonas." The presence of copper in pseudomonad and rat liver tryptophan oxygenases, previously reported by us (Brady, F. O., Monaco, M. E., Forman, H. J., Schutz, G., and Feigelson, P. (P. (1972) J. Biol. Chem. 247, 7915-7922), is reaffirmed by the experiments reported herein.     

Characteristics of substrates and inhibitors in binding to rat liver L-tryptophan 2,3-dioxygenase: a Fourier transform infrared and kinetic study.

Infrared spectroscopy and steady-state kinetics were applied to rat liver L-tryptophan 2,3-dioxygenase, in order to find relations between the structure and binding characteristics of its substrates and inhibitors. The binding characteristics were reflected by changes in the infrared CO stretch band(s) of an Fe(II)-CO complex of the enzyme upon addition of L-tryptophan and 12 analogs. The CO stretch band around 1961 cm-1 of the complex was not much affected by 1-methyl-D,L-tryptophan, a noncompetitive inhibitor, implying a binding at a site distant from the Fe(II)-CO vicinity. The spectral pattern was significantly changed by any of the other compounds which conserved an indole NH, indicative of its binding to the catalytic site. All substrates, which contained a complete CH(NH2)COOH group in addition to the NH, gave spectra similar to that of an L-tryptophan-bound complex. Spectral changes caused by six inhibitors, which lacked the complete CH(NH2)COOH, were different from one another and from those by the substrates. Hence, for an analog, the indole NH is indispensable to bind to the catalytic site, and the CH(NH2)COOH is important to take a correct configuration appropriate to the catalytic reaction. The reason why L- and D-isomers of 5-hydroxytryptohan are not substrates, in spite of their conservation of the required functional groups and correct binding to the catalytic site, has been ascribed to a possible distortion of the protein structure in the heme pocket due to a strong hydrogen bond from the hydroxyl group to an amino acid side chain.     

Functionality of biphenyl 2,3-dioxygenase components in naphthalene 1,2-dioxygenase

Naphthalene 1,2-dioxygenase (Nap dox) and biphenyl 2,3-dioxygenase (Bph dox) are related enzymes that have differentiated during evolution as their specificity has changed. Although their component arrangement is similar, the structure of each component has been modified quite extensively. The purpose of this work was to determine the catalytic capacity of purified Nap dox toward chlorobiphenyls and to investigate the functionality of Bph dox components in the Nap dox system. Both enzyme systems were purified by affinity chromatography as histidine-tagged fused proteins. Data show for the first time that Nap dox can catalyze the oxygenation of all three monochlorobiphenyl isomers, but it is unable to hydroxylate 2,5-, 2,2′-, 3,3′-, 4,4′-di- and 2,2′,5,5′-tetrachlorobiphenyl. The rates of cytochrome c reduction by the ferredoxin components of the two enzymes were identical when the Bph dox reductase component was used in the assay, showing an efficient electron transfer between the Bph dox reductase component and the Nap dox ferredoxin. However, when the Bph dox ferredoxin was used to reconstitute a hybrid Nap dox, the enzyme was only 22% as active as the parental enzyme. These data are discussed in terms of the potential use of Nap dox for the development of enhanced chlorobiphenyl-degrading dioxygenases. Received: 15 October 1998 / Received revision: 21 January 1999 / Accepted: 31 January 1999     

Utilization of dihydroflavin mononucleotide and superoxide anion for the decyclization of L-tryptophan by murine epididymal indoleamine 2,3-dioxygenase

Dihydroflavin mononucleotide (FMNH2) together with a regenerating enzyme system effectively supported L-tryptophan decyclization by indoleamine 2,3-dioxygenase isolated from murine epididymis. The native murine dioxygenase was a monomeric protein with Mr 40,000 +/- 1000, an apparent pI of 4.9 +/- 0.1, and an optimum pH within the range of 7 to 8. Using FMNH2 with FMN oxidoreductase, the enzyme attained significantly higher activity than the apparent maximal activity obtained by using the other electron donor systems examined (e.g., riboflavin, FAD, tetrahydrobiopterin, methylene blue). A kinetic study with the FMNH2 cofactor suggested the occurrence of a complex reaction (L-tryptophan-FMNH2 interdependency) and a theoretical K'm of 14 microM or a Km of 13 microM was estimated for the substrate. L-Tryptophan 2,3-dioxygenation was competitively inhibited by L-5-hydroxytryptophan with a Ki of 1 microM. The reaction rate was reduced to less than 50% of that of the control in the presence of superoxide dismutase and was decreased to 3% of the control in the absence of catalase. Thus, superoxide anion does not appear to be the only form of O2 participating in the reaction. However, these data indicate that the activation of molecular oxygen is an essential factor for an optimum catalysis and a mechanism of FMNH2-dependent oxygenation of L-tryptophan by murine indoleamine 2,3-dioxygenase.     

Tryptophan 2,3-dioxygenase: a review of the roles of the heme and copper cofactors in catalysis.

L-Tryptophan, 2,3-dioxygenase (EC 1.13.11.11) has been purified to homogenity from L-tryptophan induced Pseudomonas acidovorans (ATCC 11299b) and from L-tryptophan and cortisone induced rat liver. The enzyme from both sources is composed of four subunits and contains two g-atoms copper and two moles heme per mole tetramer. The proteins from the two sources are not identical. Three oxidation states of tryptophan oxygenase have been isolated: (1) fully oxidized, [Cu(II)]2[Ferriheme]2; (2) half reduced, [Cu(i)]2[ferriheme]2; and (3) fully reduced, [Cu(I)]2[ferroheme]2. Catalytic activity is dependent solely on the presence of Cu(I) in the enzyme, the heme may be either ferro or ferri. The presence of Cu(II) in the enzyme results in a requirement for an exogenous reductant, such as ascorbate, in order to elicit enzymic activity. Ligands, such as cyanide and carbon monoxide, can inhibit catalysis by binding to either or to both the copper and heme moieties. Metal complexing agents, such as bathocuproinesulfonate and bathophenanthrolinesulfonate, can inhibit catalysis by binding to Cu(I) resent only in catalytically active enzyme molecules. During catalysis by the fully reduced form of the enzyme, molecular oxygen binds to the heme moieties, while during catalysis by the half reduced form of the enzyme it does not, presumably binding instead to the Cu(I) moieties. Enzymes that catalyze similar reactions have been purified from other sources. Indoleamine 2,3-dioxygenase appears to be a heme protein, but its copper content is unknown. Pyrrolooxygenases appear to be completely different enzymes, although they have not yet been purified to homegeneity.     

Indoleamine 2,3-dioxygenase. Purification and some properties.

Indoleamine 2,3-dioxygenase was purified from rabbit small intestine to apparent homogeneity as judged by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The native enzyme was a monomeric protein of a molecular weight of 41,000 +/- 1,000 with an s020,w value of 3.45 S. It had a relative abundance of hydrophobic amino acids such as valine, leucine, and isoleucine, and contained approximately 5% carbohydrate by weight. The estimated content of sugar residues per mol of enzyme was: galactose, 1.2; mannose, 2.6; N-acetylglucosamine, 5.2; and sialic acid, 0.8. One mole of enzyme had 0.8 mol of protoheme IX as a prosthetic group. However, copper was not detected in a significant amount and the ratio of copper to heme was less than 0.03. EPR spectra of the nitric oxide complex of the ferrous enzyme indicated that a nitrogen atom, possibly in an imidazole group, might be coordinated as the fifth ligand of the heme coenzyme. The anisotropic g values were gx = 2.08, gy = 1.98, and gz = 2.01. A single enzyme protein catalyzed the oxygenative ring cleavage of D- and L-tryptophan, D- and L-5-hydroxytryptophan, tryptamine, and serotonin. In addition, the purified enzyme had a peroxidase activity with guaiacol and potassium iodide as hydrogen donors, but not a catalase activity.     

A myoglobin evolved from indoleamine 2,3-dioxygenase.

Hemoglobins and myoglobins are some of the best studied proteins. They are distributed in animals, plants and bacteria, and the characteristic two intron-three exon structure is widely conserved in animal globin genes (Jhiang et al., 1988). To date, all of the hemoglobins and myoglobins are believed to have a common origin, and so they are considered to be homologous. We have isolated a completely new type of myoglobin from the red muscle of the abalone Sulculus diversicolor aquatilis. The myoglobin consists of an unusual 41 kDa polypeptide chain, contains one heme per chain and forms a homodimer under physiological conditions. The cDNA-derived amino acid sequence of Sulculus myoglobin showed no significant homology with any other globins, but, surprisingly, showed high homology (35% identity) with human indoleamine 2,3-dioxygenase, a tryptophan degrading enzyme containing heme. This clearly indicates that Sulculus myoglobin evolved from a gene for indoleamine dioxygenase, but not from a globin gene. Sulculus myoglobin lacks the enzyme activity of indoleamine dioxygenase. However, in the presence of tryptophan, the autoxidation rate of oxymyoglobin was greatly accelerated, suggesting that a tryptophan binding site remains near or in the heme cavity as a relic of the molecular evolution.     

The role of serine 167 in human indoleamine 2,3-dioxygenase: a comparison with tryptophan 2,3-dioxygenase

The initial step in the l-kynurenine pathway is oxidation of l-tryptophan to N-formylkynurenine and is catalyzed by one of two heme enzymes, tryptophan 2,3-dioxygenase (TDO) or indoleamine 2,3-dioxygenase (IDO). Here, we address the role of the conserved active site Ser167 residue in human IDO (S167A and S167H variants), which is replaced with a histidine in other mammalian and bacterial TDO enzymes. Our kinetic and spectroscopic data for S167A indicate that this residue is not essential for O 2 or substrate binding, and we propose that hydrogen bond stabilization of the catalytic ferrous-oxy complex involves active site water molecules in IDO. The data for S167H show that the ferrous-oxy complex is dramatically destabilized in this variant, which is similar to the behavior observed in human TDO [Basran et al. (2008) Biochemistry 47, 4752-4760], and that this destabilization essentially destroys catalytic activity. New kinetic data for the wild-type enzyme also identify the ternary [enzyme-O 2-substrate] complex. The data reveal significant differences between the IDO and TDO enzymes, and the implications of these results are discussed in terms of our current understanding of IDO and TDO catalysis.     

Comparative effects of oxygen on indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase of the kynurenine pathway

Indoleamine 2,3-dioxygenase (IDO) reacts with either oxygen or superoxide and tryptophan (trp) or other indoleamines while tryptophan 2,3-dioxygenase (TDO) reacts with oxygen and is specific for trp. These enzymes catalyze the rate-limiting step in the kynurenine (KYN) pathway from trp to quinolinic acid (QA) with TDO in kidney and liver and IDO in many tissues, including brain where it is low but inducible. QA, which does not cross the blood-brain barrier, is an excitotoxin found in the CNS during various pathologies and is associated with convulsions. We proposed that HBO-induced convulsions result from increased flux through the KYN pathway via oxygen stimulation of IDO. To test this, TDO and IDO of liver and brain, respectively, of Sprague Dawley rats were assayed with oxygen from 0 to 6.2 atm HBO. TDO activity was appreciable at even 30 microM oxygen and rose steeply to a maximum at 40 microM. Conversely, IDO had almost no detectable activity at or below 100 microM oxygen and maximum activity was not reached until about 1150 microM. (Plasma contains about 215 microM oxygen and capillaries about 20 microM oxygen when rats breathe air.) KYN was 60% higher in brains of HBO-convulsed rats compared to rats breathing air. While the oxygen concentration inside cells of rats breathing air or HBO is not known precisely, it is clear that the rate-limiting, IDO-catalyzed step in the brain KYN pathway (but not liver TDO) can be greatly accelerated in rats breathing HBO.