Purification and characterization of tryptophan dioxygenase from Streptomyces parvulus

Tryptophan dioxygenase, derived from Streptomyces parvulus, was purified to near homogeneity and shown to have a native Mr of 88,000. Kinetic parameters of the enzyme were determined and evidence suggesting that it is a hemoprotein was obtained. Tryptophan dioxygenase has a high specificity toward L-tryptophan with an apparent Km of 0.3 mM. L-3-Hydroxykynurenine was a competitive inhibitor with respect to L-tryptophan with a Ki of 0.16 mM. In vitro, the enzyme displayed little activity in the absence of a reducing agent; ascorbate, at 50 mM, was the preferred reductant providing almost a 50-fold increase in enzyme activity. The regulation of tryptophan dioxygenase synthesis and activity is described. The expression of the enzyme is correlated with the biosynthesis of actinomycin D in S. parvulus. These results support the hypothesis that tryptophan dioxygenase functions as the first enzyme in the sequence converting L-tryptophan to the chromophore of this antibiotic.     

Single crystal EPR of myoglobin nitroxide. Freezing-induced reversible changes in the molecular orientation of the ligand

Single crystals of myoglobin nitroxide (MbNO) are examined by the electron paramagnetic resonance spectroscopy at ambient and cryogenic temperatures for both the 14NO and 15NO derivatives. The principal values and the eigenvectors of the g tensor and the hyperfine coupling tensor are determined: g xx = 2.050, g yy = 2.022, and g zz = 1.993; A xi xi = 15.6, A zeta zeta = 21.4, and A eta eta = 26.7 G for the nitrogen in 15NO at ambient temperature. The Fe--N--O bond angle is calculated to be 153 degrees. This result is in good agreement with the x-ray structural result on the six-liganded model compound with the bent Fe--N--O configuration. The principal values and the eigenvectors of the g tensor and the hyperfine coupling tensor are also determined at 77 K for Mb15NO; gxx = 2.076, gyy = 1.979, and gzz = 2.002; A xi xi = 21, A zeta zeta = 24, and A eta eta = 27 G. The Fe--N--O bond angle is calculated to be 109 degrees. The hyperfine splittings attributed to N epsilon atom of proximal histidine are observed in the direction of the gzz at both temperatures. The drastic shift of the EPR spectrum of MbNO single crystal is observed below the freezing point of this crystal. It clearly demonstrates that the conformation of the bonding NO is drastically altered upon freezing. The temperature dependence of the EPR spectra of MbNO below the freezing point cannot be explained only by appropriate combinations of the higher temperature type and the lower temperature type and suggests the contribution from an unpaired electron with the iron dz2 and dyz (or dxz) orbitals. The present EPR results demonstrated that the changes in the molecular orientations are induced by freezing of the biological molecules without disorder of the crystal lattice.     

Spectroscopic evidence for a copper-nitrosyl intermediate in nitrite reduction by blue copper-containing nitrite reductase

The reactions of nitrogen monoxide (NO) with the blue copper-containing nitrite reductases from Alcaligenes sp. NCIB 11015 and Achromobacter cycloclastes IAM 1013 were investigated spectroscopically. The electron paramagnetic resonance (EPR) signals of the blue coppers vanished in the presence of NO at 77 K, being fully restored by the removal of NO. The additions of NO to the enzyme solutions resulted in the substantial bleaching of the visible absorption bands at room temperature. The reactions were also completely reversible. These results suggest the formation of a cuprous nitrosyl complex (Cu+-NO+), which is likely the intermediate in the enzymatic nitrite reduction.     

Electron paramagnetic resonance spectroscopy of lactoperoxidase complexes: clarification of hyperfine splitting for the NO adduct of lactoperoxidase

Electron paramagnetic resonance (EPR) studies of the nitrosyl adduct of ferrous lactoperoxidase (LPO) confirm that the fifth axial ligand in LPO is bound to the iron via a nitrogen atom. Complete reduction of the ferric LPO sample is required in order to observe the nine-line hyperfine splitting in the ferrous LPO/NO EPR spectrum. The ferrous LPO/NO complex does not exhibit a pH or buffer system dependence when examined by EPR. Interconversion of the ferrous LPO/NO complex and the ferric LPO/NO2- complex is achieved by addition of the appropriate oxidizing or reducing agent. Characterization of the low-spin LPO/NO2- complex by EPR and visible spectroscopy is reported. The pH dependence of the EPR spectra of ferric LPO and ferric LPO/CN- suggests that a high-spin anisotropic LPO complex is formed at high pH and an acid-alkaline transition of the protein conformation near the heme site does occur in LPO/CN-. The effect of tris(hydroxymethyl)aminomethane buffer on the LPO EPR spectrum is also examined.     

Type 2 Cu2+ in pMMO from Methylomicrobium album BG8

EPR spectra were obtained for the type 2 Cu2+ site in particulate methane monooxygenase (pMMO) from Methylomicrobium album BG8 grown on K15NO3 and 63Cu(NO3)2. The concentration of the type 2 Cu2+ signal was approximately 200 microM per 25 mg/ml protein in packed cells and membrane fractions, a concentration that is consistent with its attribution to pMMO, and the EPR parameters were consistent with electron paramagnetic resonance (EPR) parameters previously assigned to pMMO. The superhyperfine structure due to nitrogen is better resolved because I = 1/2 for 15N whereas I = 1 for 14N and A(15N)/A(14N) = 1.4. Under these conditions, superhyperfine structure is resolved in the g region of the X-band spectrum. At low microwave frequency (S-band) the resolution of the nitrogen superhyperfine structure improves. Signals are attributed to type 2 Cu2+ in which cupric ion is bound to four (less likely three) nitrogen donor atoms.     

Biochemical and medical aspects of the indoleamine 2,3-dioxygenase-initiated L-tryptophan metabolism

Indoleamine 2,3-dioxygenase (EC 1.13.11.42) is a heme-containing dioxygenase which catalyzes the first and rate-limiting step in the major pathway of L-tryptophan catabolism in mammals. Much attention has recently been focused on the dioxygenase because this metabolic pathway is involved not only in a variety of physiological functions but also in many diseases. In this review, the discovery and unique catalytic properties of dioxygenase are described first, and then the recent findings regarding the dioxygenase-initiated tryptophan metabolism are summarized, with special emphasis on the detrimental role of dioxygenase in side effects of interferon-gamma and interleukin-12 (by systemic tryptophan depletion), the escape of malignant tumors from immune surveillance (by immunosuppression caused by tryptophan depletion), several neurodegenerative disorders including Alzheimer's disease (by an aberrant production of neurotoxin, quinolinic acid), and age-related cataract (due to "Kynurenilation," a novel post-translational modification of lens proteins with tryptophan-derived UV filters).     

Characterization of the radical product formed from the reaction of nitric oxide with the spin trap 3,5-dibromo-4-nitrosobenzene sulfonate.

Previously, 3,5-dibromo-4-nitrosobenzene sulfonate (DBNBS) has been used in combination with electron paramagnetic resonance (EPR) spectrometry to trap nitric oxide (NO(*)). The reaction between DBNBS and NO(*) yields a radical product which gives rise to an EPR signal consisting of three lines with an A(N) = 0.96 mT, but the structure of this product is unknown. A two-stage high-performance liquid chromatography fractionation was performed to isolate the radical product from the other components in the DBNBS/NO(*) reaction mixture. The fractions containing the radical product were identified by the presence of the three-line EPR signal, and then these fractions were analyzed by negative ion fast atom bombardment-mass spectrometry (FAB-MS). Collectively, the FAB-MS data suggested that the radical product is the monosodium electrostatic complex with the dianion, bis(2,6-dibromo-4-sulfophenyl) nitroxyl. Analysis of the Gaussian and Lorentzian linewidths of the EPR signal suggested that bis(2,6-dibromo-4-sulfophenyl) nitroxyl molecules may group together to form micelles. Further studies also indicated that significant amounts of nitrogen and nitrate were produced during the reaction between DBNBS and NO(*). A reaction scheme consistent with these results is presented.     

Interaction of bacteriophage lambda protein phosphatase with Mn(II): evidence for the formation of a [Mn(II)]2 cluster.

The interaction of bacteriophage lambda protein phosphatase with Mn2+ was studied using biochemical techniques and electron paramagnetic resonance spectrometry. Reconstitution of bacteriophage lambda protein phosphatase in the presence of excess MnCl2 followed by rapid desalting over a gel filtration column resulted in the retention of approximately 1 equiv of Mn2+ ion bound to the protein. This was determined by metal analyses and low-temperature EPR spectrometry, the latter of which provided evidence of a mononuclear high-spin Mn2+ ion in a ligand environment of oxygen and nitrogen atoms. The Mn2+-reconstituted enzyme exhibited negligible phosphatase activity in the absence of added MnCl2. The EPR spectrum of the mononuclear species disappeared upon the addition of a second equivalent of Mn2+ and was replaced by a spectrum attributed to an exchange-coupled (Mn2+)2 cluster. EPR spectra of the dinuclear (Mn2+)2 cluster were characterized by the presence of multiline features with a hyperfine splitting of 39 G. Temperature-dependent studies indicated that these features arose from an excited state. Titrations of the apoprotein with MnCl2 provided evidence of one Mn2+ binding site with a micromolar affinity and at least one additional Mn2+ site with a 100-fold lower affinity. The dependence of the phosphatase activity on Mn2+ concentration indicates that full enzyme activity probably requires occupation of both Mn2+ sites. These results are discussed in the context of divalent metal ion activation of this enzyme and possible roles for Mn2+ activation of other serine/threonine protein phosphatases.     

Oxygenation and EPR spectral properties of Aplysia myoglobins containing cobaltous porphyrins.

Cobalt myoglobins (Aplysia) have been reconstituted from apo-myoglobin (Aplysia) and proto-, meso-, and deutero-cobalt porphyrins. Each of them showed the 30--60 times lower oxygen affinity than those of the corresponding cobalt myoglobins (Sperm whale). Kinetic investigation of their oxygenation by the temperature-junp relaxation technique showed that the low oxygen affinity of cobalt myoglobin (Aplysia) is due to a large dissociation rate constant. the electron paramagnetic resonance (EPR) spectrum of oxy cobalt myoglobin (Aplysia) is affected by the replacement of H2O with D2O, suggesting a possible interaction between the bound oxygen and the neighboring hydrogen atom. A low temperature photodissociation study showed that the product of photolysis of oxy cobalt myoglobin (Aplysia) gives an EPR spectrum different from that of the deoxy-cobalt myoglobin (Aplysia) and from that of the photolysed form of oxy-cobalt myogloin (Sperm whale). These observations suggest that in oxy-cobalt myoglobin (Aplysia) the bound oxygen might interact with amino acid adjacent to it, but the interaction is weaker than that in oxy cobalt myoglobin (Sperm whale).     

Role of the tryptophan residue in the interaction of pancreozymin with its receptor

1. Analogues of the C-terminal octapeptide and tetrapeptide of pancreozymin with a modified tryptophan residue have been tested on the rat pancreas adenylate cyclase activity, on the enzyme and fluid secretion of the rat pancreas in vivo and on the amylase release from rabbit pancreatic fragments. 2. Fluorination of the tryptophan residue in position 5 or 6 does not influence the effect of the peptides on any of the measured parameters. 3. Methylation of the nitrogen atom in the indolyl ring, which eliminates hydrogen bond formation, markedly reduces the affinity of the peptides for the adenylate cyclase activity and for the amylase release in rabbit pancreatic fragments. The effects on fluid and enzyme secretion in the rat pancreas in vivo are reduced nearly as much. 4. Tetrafluorination of the tryptophan residue, which reduces its charge donor capacity, causes a still larger reduction in activity and affinity of the octapeptide. 5. The tetrafluorinated tetrapeptide stimulates the adenylate cyclase activity and the enzyme and fluid secretion in vivo more than the unmodified tetrapeptide, which may be due to its increased hydrophobicity. 6. Replacement of the nitrogen atom in the indolyl ring of tryptophan by a sulfur or an oxygen atom, which also reduces the charge donor capacity, leads again to a large reduction in the affinity and activity of both the octapeptide and the tetrapeptide. 7. These findings suggest that the charge donor capacity of the tryptophan residue is of primary importance for the biologic activity of pancreozymin, while hydrogen bond formation and hydrophobicity are of secondary importance.     

Electron paramagnetic resonance study of nitrosylprotoheme dimethyl ester complexes with aliphatic nitrogenous bases: characterization of the axial ligand trans to the nitrosyl group in nitrosylhemoproteins

The interaction of nitrosyl(protoporphyrin IX dimethyl ester) iron (II) (Fe(PPDME)(NO)) with aliphatic amines, anilines, and cyclic imines has been studied by electron paramagnetic resonance (EPR) measurements at room temperature and at 77K. At room temperature, the bases studied here were divided into two groups according to the exchange rate between two EPR-positive species, Fe(PPDME)(NO) and Fe(PPDME)(NO)B, in equilibrium; Fe(PPDME)(NO) + B

Fe(PPDME)-(NO)B. The Fe(PPDME)(NO)-base system with a fast exchange rate on the EPR time scale had a smaller equilibrium constant (K) than that with a slow rate. The EPR spectra of the Fe(PPDME)(NO)-base system both at room temperature and at 77K were markedly influenced by the steric interaction of the base with the porphyrin core.     

Biochemical,Kinetic, and Spectroscopic Characterization of Ruegeria pomeroyi DddW—A Mononuclear Iron-Dependent DMSP Lyase

The osmolyte dimethylsulfoniopropionate (DMSP) is a key nutrient in marine environments and its catabolism by bacteria through enzymes known as DMSP lyases generates dimethylsulfide (DMS), a gas of importance in climate regulation, the sulfur cycle, and signaling to higher organisms. Despite the environmental significance of DMSP lyases, little is known about how they function at the mechanistic level. In this study we biochemically characterize DddW, a DMSP lyase from the model roseobacter Ruegeria pomeroyi DSS-3. DddW is a 16.9 kDa enzyme that contains a C-terminal cupin domain and liberates acrylate, a proton, and DMS from the DMSP substrate. Our studies show that as-purified DddW is a metalloenzyme, like the DddQ and DddP DMSP lyases, but contains an iron cofactor. The metal cofactor is essential for DddW DMSP lyase activity since addition of the metal chelator EDTA abolishes its enzymatic activity, as do substitution mutations of key metal-binding residues in the cupin motif (His81, His83, Glu87, and His121). Measurements of metal binding affinity and catalytic activity indicate that Fe(II) is most likely the preferred catalytic metal ion with a nanomolar binding affinity. Stoichiometry studies suggest DddW requires one Fe(II) per monomer. Electronic absorption and electron paramagnetic resonance (EPR) studies show an interaction between NO and Fe(II)-DddW, with NO binding to the EPR silent Fe(II) site giving rise to an EPR active species (g = 4.29, 3.95, 2.00). The change in the rhombicity of the EPR signal is observed in the presence of DMSP, indicating that substrate binds to the iron site without displacing bound NO. This work provides insight into the mechanism of DMSP cleavage catalyzed by DddW.     

Enhancement of Tryptophan Uptake by Divalent Cations in the Absence of Sodium Ions

Abstract: Nations were found to inhibit the uptake of L-tryptophan into synaptosomes with a shallow dose-response curve. Almost maximal inhibition was obtained with 10 mM-Na⁺. The divalent cations Ca²⁺ and Mg²⁺ were shown to be responsible for the increased uptake of L-tryptophan in the absence of Na⁺ ions. Other divalent cations also promoted tryptophan uptake under this condition (Ca²⁺ < Mg²⁺ < Mn²⁺ < Fe²⁺ < Zn²⁺ < Cu²⁺). It was concluded that monovalent chelate complexes were responsible for this enhancing effect. The measured L-tryptophan uptake was the net product of membrane bound and unbound tryptophan. Both bound and unbound tryptophan were increased in the presence of divalent cations. If no divalent cations were added to the incubation medium, Na⁺ ions decreased the unbound tryptophan but were without effect on bound tryptophan. Under these circumstances D-tryptophan had no effect on binding of the L-isomer and affected the transport of 1.-tryptophan only at very high does (100 x conc. L-tryptophan). These results suggest that I -tryptophan binds to a stereospecific transport carrier located in the synaptosomal membrane and that Na⁺ ions prevent the translocation of this carrier amino acid complex from the outer to the inner site of the neuronal membrane.     

Interactions of tryptophan synthase, tryptophanase, and pyridoxal phosphate with oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan: support for an indolenine intermediate in tryptophan metabolism

We have examined the interaction of tryptophan synthase and tryptophanase with the tryptophan analogues oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan. Since these analogues have tetrahedral geometry at carbon 3 of the heterocyclic ring, they are structurally similar to the indolenine tautomer of L-tryptophan, a proposed intermediate in reactions of L-tryptophan. Oxindolyl-L-alanine and 2,3-dihydro-L-tryptophan are potent competitive inhibitors of both tryptophan synthase and tryptophanase, with KI values (3-17 microM) 10-100-fold lower than the corresponding Km or KI values for L-tryptophan. Addition of oxindolyl-L-alanine or 2,3-dihydro-L-tryptophan to solutions of the alpha 2 beta 2 complex of tryptophan synthase results in new absorption bands at 480 or 494 nm, respectively, which are ascribed to a quinonoid or alpha-carbanion intermediate. Spectrophotometric titration data give half-saturation values of 5 and 25 microM, which are comparable to the KI values obtained in kinetic experiments. Our finding that both enzymes catalyze incorporation of tritium from 3H2O into oxindolyl-L-alanine is evidence that both enzymes form alpha-carbanion intermediates with oxindolyl-L-alanine. These results support the proposal that the indolenine tautomer of L-tryptophan is an intermediate in reactions catalyzed by both tryptophanase and tryptophan synthase. In addition, we have found that oxindolyl-L-alanine reacts irreversibly with free pyridoxal phosphate to form a covalent adduct.     

Effects of mutations in the L-tryptophan binding pocket of the Trp RNA-binding attenuation protein of Bacillus subtilis

The Bacillus subtilis tryptophan biosynthetic genes are regulated by the trp RNA-binding attenuation protein (TRAP). Cooperative binding of L-tryptophan activates TRAP so that it can bind to RNA. The crystal structure revealed that L-tryptophan forms nine hydrogen bonds with various amino acid residues of TRAP. We performed site-directed mutagenesis to determine the importance of several of these hydrogen bonds in TRAP activation. We tested both alanine substitutions as well as substitutions more closely related to the natural amino acid at appropriate positions. Tryptophan binding mutations were identified in vivo having unchanged, reduced, or completely eliminated repression activity. Several of the in vivo defective TRAP mutants exhibited reduced affinity for tryptophan in vitro but did not interfere with RNA binding at saturating tryptophan concentrations. However, a 10-fold decrease in TRAP affinity for tryptophan led to an almost complete loss of regulation, whereas increased TRAP affinity for tryptophan had little or no effect on the in vivo regulatory activity of TRAP. One hydrogen bond was found to be dispensable for TRAP activity, whereas two others appear to be essential for TRAP function. Another mutant protein exhibited tryptophan-independent RNA binding activity. We also found that trp leader RNA increases the affinity of TRAP for tryptophan.     

The interaction of nitric oxide with soybean lipoxygenase-1.

The interaction of nitric oxide with the non-heme iron dioxygenase lipoxygenase is reported. This apparently resulted in a novel type of complex where an electron is donated to the NO molecule. In addition a new position for an EPR transition from iron was discovered which, it is suggested results from high spin ferric iron in a field of axial symmetry characterised by a very low value for D.     

The actions of interferon and antiinflammatory agents of induction of indoleamine 2,3-dioxygenase in human peripheral blood monocytes

Interferon substantially induced indoleamine 2,3-dioxygenase and increased L-tryptophan metabolism in human peripheral blood monocytes. The induction of dioxygenase by gamma-interferon was significantly higher than that observed with alpha-interferon. This cytokine-dependent induction of the enzyme was markedly and differentially altered by antiinflammatory drugs (i.e., acetaminophen, 3-deazaadenosine, indomethacin and dexamethasone). Dexamethasone potentiated the effect of gamma-interferon and resulted in "super-induction" of the enzyme. This is the first demonstration of the interferon-elicited induction of the dioxygenase in the cells of the immune system and of a novel mechanism for regulating tryptophan metabolism in the cells.     

Sources and implications of basal nitric oxide in spontaneous contractions of guinea pig taenia caeci

We examined in vitro the source and role of basal nitric oxide (NO) in proximal segments of guinea pig taenia caeci in nonadrenergic, noncholinergic (NANC) conditions. Using electron paramagnetic resonance (EPR), we measured the effect of the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), the neuronal blocker tetrodotoxin (TTX, 10(-6) M), or both on spontaneous contractions and on the production of basal NO. Both L-NAME and TTX, when tested alone, increased the amplitude and frequency of contractions. NO production was abolished by L-NAME and was inhibited by 38% by TTX. When tested together, L-NAME in the presence of TTX or TTX in the presence of L-NAME had no further effect on the amplitude or frequency of spontaneous contractions, and the NO production was inhibited. These findings suggest that basal NO consists of TTX-sensitive and TTX-resistant components. The TTX-sensitive NO has an inhibitory effect on spontaneous contractions; the role of TTX-resistant NO is unknown.     

The structural basis for the interaction between L-tryptophan and the Escherichia coli trp aporepressor

We have employed equilibrium dialysis to help study the mechanism by which the unliganded Escherichia coli trp aporepressor is activated by L-tryptophan to the liganded trp repressor. By measuring the relative affinity of L-tryptophan and various tryptophan analogues for the co-repressor's binding site, we have estimated the extent to which each of the functional groups of L-tryptophan contributes to the liganding process and discuss their role in the context of the crystal structures of the trp repressor and aporepressor. We have found that the indole ring and alpha carboxyl group of L-tryptophan are mainly responsible for its affinity to the aporepressor. The alpha amino group, however, has a small negative contribution to the affinity of L-tryptophan for the aporepressor which may be associated with its essential role in operator-specific binding.     

Dinitrosyl-iron complexes with thiol-containing ligands: spatial and electronic structures.

Parameters of the EPR signals of monomeric dinitrosyl-iron complexes with 1H-1,2,4-triazole-3-thiol (DNIC-MT), obtained by treating MT+ferrous iron in DMSO solution with gaseous NO, have been compared with those of the crystalline monomeric DNIC-MT with tetrahedral structure. Dissolved DNIC-MT were characterized by the isotropic EPR signal centered at g=2.03 with half-width of 0.7 mT and quintet hyperfine structure when recorded at ambient temperature or the anisotropic EPR signal with g( perpendicular)=2.045, g( parallel)=2.014 from frozen solution at 77 kappa, Cyrillic. DNIC-MT in crystalline state showed the structure-less symmetrical singlet EPR signal centered at g=2.03 and half-width of 1.7 mT at both room and liquid nitrogen temperature. The Lorentz shape of this signal indicates the strong exchange interaction between these complexes in the DNIC-MT crystal. Being dissolved in DMSO the crystalline sample of DNIC-MT demonstrated the EPR signal typical for DNIC-MT, obtained by treating MT+ferrous iron in DMSO solution with gaseous NO. Low spin (S=1/2) d(9) electron configuration of DNIC-MT with tetrahedral structure (formula [(MT-S(.))(2)Fe(-1)(NO(+))(2)](+)) was suggested to be responsible for the signal of DNIC-MT in crystalline state. Dissolving of the crystals of DNIC-MT may result in the change of their spatial and electronic structure, namely, tetrahedral structure of the complexes characterized by low spin d(9) electronic configuration transforms into a plane-square structure with d(7) electronic configuration and low spin S=1/2 state (formula [(MT- S(-))(2)Fe(+)(NO(+))(2)](+)). The latter was suggested to be characteristic of other DNICs with various thiol-containing ligands in the solutions. The proposed mechanism of these DNICs formation from ferrous iron, thiol and NO shows that the process could be accompanied by the ionization of NO molecules to NO(+) and NO(-) ions in the complexes. Detailed analysis of the shape of the EPR signals of these complexes provided additional information about the exchange interaction typical for DNIC-MT in crystals.