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Regioselective Nitration of Tryptophan by a Complex between Bacterial Nitric-oxide Synthase and Tryptophanyl-tRNA Synthetase

2004; Elsevier BV; Volume: 279; Issue: 48 Linguagem: Inglês

10.1074/jbc.c400418200

ISSN

1083-351X

Autores

Madhavan R. Buddha, Tao Tao, Ronald J. Parry, Brian R. Crane,

Tópico(s)

Metabolomics and Mass Spectrometry Studies

Resumo

Bacterial nitric-oxide synthase proteins (NOSs) from certain Streptomyces strains have been shown to participate in biosynthetic nitration of tryptophanyl moieties in vivo (Kers, J. A., Wach, M. J., Krasnoff, S. B., Cameron, K. D., Widom, J., Bukhaid, R. A., Gibson, D. M., and Crane, B. R., and Loria, R. (2004) Nature 429, 79–82). We report that the complex between Deinococcus radiodurans NOS (deiNOS) and an unusual tryptophanyl-tRNA synthetase (TrpRS II) catalyzes the regioselective nitration of tryptophan (Trp) at the 4-position. Unlike non-enzymatic Trp nitration, and similar reactions catalyzed by globins and peroxidases, deiNOS only produces the otherwise unfavorable 4-nitro-Trp isomer. Although deiNOS alone will catalyze 4-nitro-Trp production, yields are significantly enhanced by TrpRS II and ATP. 4-Nitro-Trp formation exhibits saturation behavior with Trp (but not tyrosine) and is completely inhibited by the addition of the mammalian NOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (H4B). Trp stimulates deiNOS oxidation of substrate l-arginine (Arg) to the same degree as H4B. These observations are consistent with a mechanism where Trp or a derivative thereof binds in the NOS pterin site, participates in Arg oxidation, and becomes nitrated at the 4-position. Bacterial nitric-oxide synthase proteins (NOSs) from certain Streptomyces strains have been shown to participate in biosynthetic nitration of tryptophanyl moieties in vivo (Kers, J. A., Wach, M. J., Krasnoff, S. B., Cameron, K. D., Widom, J., Bukhaid, R. A., Gibson, D. M., and Crane, B. R., and Loria, R. (2004) Nature 429, 79–82). We report that the complex between Deinococcus radiodurans NOS (deiNOS) and an unusual tryptophanyl-tRNA synthetase (TrpRS II) catalyzes the regioselective nitration of tryptophan (Trp) at the 4-position. Unlike non-enzymatic Trp nitration, and similar reactions catalyzed by globins and peroxidases, deiNOS only produces the otherwise unfavorable 4-nitro-Trp isomer. Although deiNOS alone will catalyze 4-nitro-Trp production, yields are significantly enhanced by TrpRS II and ATP. 4-Nitro-Trp formation exhibits saturation behavior with Trp (but not tyrosine) and is completely inhibited by the addition of the mammalian NOS cofactor (6R)-5,6,7,8-tetrahydro-l-biopterin (H4B). Trp stimulates deiNOS oxidation of substrate l-arginine (Arg) to the same degree as H4B. These observations are consistent with a mechanism where Trp or a derivative thereof binds in the NOS pterin site, participates in Arg oxidation, and becomes nitrated at the 4-position. The generation and fate of reactive nitrogen species (RNS) 1The abbreviations used are: RNS, reactive nitrogen species; Arg, l -arginine; Cit, l -citrulline, NHA, Nω-hydroxyl-l -arginine, NOS, nitricoxide synthase; mNOS, mammalian NOS; deiNOS, D. radiodurans NOS; NOSred, NOS reductase domain; NOSox, NOS oxygenase domain; iNOS, inducible NOS; H4B, (6R)-5,6,7,8-tetrahydro-l -biopterin; NO, nitric oxide; [NO], nitric oxide, nitroxyl (NO–), or nitrosium (NO+); SB, solubilizing buffer; Trp, l -tryptophan; TrpRS II, the non-standard tryptophanyl tRNA synthetase of D. radiodurans.1The abbreviations used are: RNS, reactive nitrogen species; Arg, l -arginine; Cit, l -citrulline, NHA, Nω-hydroxyl-l -arginine, NOS, nitricoxide synthase; mNOS, mammalian NOS; deiNOS, D. radiodurans NOS; NOSred, NOS reductase domain; NOSox, NOS oxygenase domain; iNOS, inducible NOS; H4B, (6R)-5,6,7,8-tetrahydro-l -biopterin; NO, nitric oxide; [NO], nitric oxide, nitroxyl (NO–), or nitrosium (NO+); SB, solubilizing buffer; Trp, l -tryptophan; TrpRS II, the non-standard tryptophanyl tRNA synthetase of D. radiodurans. are important to a variety of physiological processes that include pathogen cell death, general progression toward disease states, and stimulation of regulatory pathways (1Beckman J.S. Chem. Res. Toxicol. 1996; 9: 836-844Crossref PubMed Scopus (911) Google Scholar, 2Ischiropoulos H. Free Radic. Biol. Med. 2002; 33: 727-874Crossref Scopus (2) Google Scholar, 3Eiserich J.P. Baldus S. Brennan M.L. Ma W.X. Zhang C.X. Tousson A. Castro L. Lusis A.J. Nauseef W.M. White C.R. Freeman B.A. Science. 2002; 296: 2391-2394Crossref PubMed Scopus (591) Google Scholar). For example, nitrated aromatic protein residues mark the involvement of RNS in neurodegenerative diseases, artherosclereosis, infections, inflammation, and cancer (1Beckman J.S. Chem. Res. Toxicol. 1996; 9: 836-844Crossref PubMed Scopus (911) Google Scholar, 2Ischiropoulos H. Free Radic. Biol. Med. 2002; 33: 727-874Crossref Scopus (2) Google Scholar, 3Eiserich J.P. Baldus S. Brennan M.L. Ma W.X. Zhang C.X. Tousson A. Castro L. Lusis A.J. Nauseef W.M. White C.R. Freeman B.A. Science. 2002; 296: 2391-2394Crossref PubMed Scopus (591) Google Scholar). Globins and peroxidases have been shown to catalyze complex reactions that can result in nitration of aromatic amino acids (3Eiserich J.P. Baldus S. Brennan M.L. Ma W.X. Zhang C.X. Tousson A. Castro L. Lusis A.J. Nauseef W.M. White C.R. Freeman B.A. Science. 2002; 296: 2391-2394Crossref PubMed Scopus (591) Google Scholar, 4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar, 5Herold S. Shivashankar K. Mehl M. Biochemistry. 2002; 41: 13460-13472Crossref PubMed Scopus (62) Google Scholar, 6Herold S. Free Radic. Biol. Med. 2004; 36: 565-579Crossref PubMed Scopus (53) Google Scholar, 7Brennan M.L. Wu W.J. Fu X.M. Shen Z.Z. Song W. Frost H. Vadseth C. Narine L. Lenkiewicz E. Borchers M.T. Lusis A.J. Lee J.J. Lee N.A. Abu-Soud H.M. Ischiropoulos H. Hazen S.L. J. Biol. Chem. 2002; 277: 17415-17427Abstract Full Text Full Text PDF PubMed Scopus (454) Google Scholar). In mammals, a primary source of RNS is nitric oxide (NO), which is largely produced by the three isozymes of nitric-oxide synthase (NOS) (8Stuehr D.J. Santolini J. Wang Z. Wei C. Adak S. J. Biol. Chem. 2004; 279: 36167-36170Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar). Whereas mammalian NOS-mediated nitration events are generally nonspecific, a bacterial nitric-oxide synthase protein has recently been shown to participate in a specific biosynthetic nitration reaction (9Kers J.A. Wach M.J. Krasnoff S.B. Widom J. Cameron K.D. Bukhalid R.A. Gibson D.M. Crane B.R. Loria R. Nature. 2004; 429: 79-82Crossref PubMed Scopus (205) Google Scholar). Some pathogenic Streptomyces produce a family of unusual phytotoxin dipeptides (derivatives of cyclo-(l-tryptophanyl-l-phenylalanyl)) called thaxtomins that contain a 4-nitro-tryptophanyl moiety (10Healy F.G. Wach M. Krasnoff S.B. Gibson D.M. Loria R. Mol. Microbiol. 2000; 38: 794-804Crossref PubMed Scopus (169) Google Scholar). The transferable pathogenicity island that contains the genes responsible for thaxtomin biosynthesis also codes for a NOS (9Kers J.A. Wach M.J. Krasnoff S.B. Widom J. Cameron K.D. Bukhalid R.A. Gibson D.M. Crane B.R. Loria R. Nature. 2004; 429: 79-82Crossref PubMed Scopus (205) Google Scholar). Genetic and isotope labeling studies have shown that the Streptomyces NOS participates in thaxtomin nitration (9Kers J.A. Wach M.J. Krasnoff S.B. Widom J. Cameron K.D. Bukhalid R.A. Gibson D.M. Crane B.R. Loria R. Nature. 2004; 429: 79-82Crossref PubMed Scopus (205) Google Scholar). The chemical mechanism of a NOS-mediated nitration is intriguing because NO is unlikely to react directly wiith indole (11Koppenol W.H. Free Radic. Biol. Med. 1998; 25: 385-391Crossref PubMed Scopus (303) Google Scholar, 12Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Crossref PubMed Scopus (242) Google Scholar). Another link between tryptophan metabolism and bacterial NOS comes from our recent finding that in Deinococcus radiodurans, an unusual tryptophanyl-tRNA synthetase (TrpRS II) interacts with the D. radiodurans NOS (deiNOS) (31Buddha M.R. Keery K. Crane B.R. Proc. Natl. Acad. Sci. 2004; (U. S. A., in press)PubMed Google Scholar). In addition to catalyzing charging of tRNATrp, TrpRS II increases NOS solubility, affinity for substrate, and NO-synthase activity (31Buddha M.R. Keery K. Crane B.R. Proc. Natl. Acad. Sci. 2004; (U. S. A., in press)PubMed Google Scholar). Uncharacteristically, D. radiodurans has two TrpRS genes: the first has ∼40% identity to other typical TrpRS sequences, and the second, which interacts with NOS, has only ∼28% identity to other TrpRS sequences. Type I tRNA synthetases catalyze two reactions in the production of aminoacyl-tRNA (13Doublie S. Bricogne G. Gilmore C. Carter C.W. Structure (Lond.). 1995; 3: 17-31Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). First, the amino acid is activated to the aminoacyl-5′-adenylate, which is then reacted with the 3′-acceptor stem of tRNA to form the aminoacyl-tRNA. Adenylation of amino acids also precedes their condensation into natural products by non-ribosomal peptide synthases, such as the enzymes that produce thaxtomin (10Healy F.G. Wach M. Krasnoff S.B. Gibson D.M. Loria R. Mol. Microbiol. 2000; 38: 794-804Crossref PubMed Scopus (169) Google Scholar). Thus, deiNOS and stNOS both associate, either physically or genetically, with enzymes capable of activating Trp for subsequent chemical coupling. Bacterial NOSs are homologous to the mammalian oxygenase domain (mNOSox) but lack an associated reductase domain (NOSred) (14Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar, 15Pant K. Bilwes A.M. Adak S. Stuehr D.J. Crane B.R. Biochemistry. 2002; 41: 11071-11079Crossref PubMed Scopus (122) Google Scholar, 16Bird L.E. Ren J.S. Zhang J.C. Foxwell N. Hawkins A.R. Charles I.G. Stammers D.K. Structure (Lond.). 2002; 10: 1687-1696Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Adak S. Aulak K. Stuehr D.J. J. Biol. Chem. 2002; 277: 16167-16171Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). Compared with mNOSs, bacterial NOSs have many similar physical, chemical, and structural properties that include that ability to catalyze the formation of nitric oxide from Arg in a manner dependent on reduced pterins (either with (6R)-5,6,7,8-tetrahydro-l-biopterin (H4B) or with the related cofactor tetrahydrofolate (14Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar, 15Pant K. Bilwes A.M. Adak S. Stuehr D.J. Crane B.R. Biochemistry. 2002; 41: 11071-11079Crossref PubMed Scopus (122) Google Scholar, 16Bird L.E. Ren J.S. Zhang J.C. Foxwell N. Hawkins A.R. Charles I.G. Stammers D.K. Structure (Lond.). 2002; 10: 1687-1696Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 17Adak S. Aulak K. Stuehr D.J. J. Biol. Chem. 2002; 277: 16167-16171Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar)). In mNOS, H4B functions to rapidly supply an electron to the heme center during oxidation activation for conversion of Arg to the intermediate Nω-hydroxyl-l-arginine (NHA) and for subsequent oxidation of NHA to citrulline and NO (8Stuehr D.J. Santolini J. Wang Z. Wei C. Adak S. J. Biol. Chem. 2004; 279: 36167-36170Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 18Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (166) Google Scholar). Herein, we report that the deiNOS-TrpRS II complex catalyzes the regioselective nitration of Trp at the 4-position. Un-like non-enzymatic nitration of Trp, as well as those reactions catalyzed by peroxidases and globins, no other nitrated Trp byproducts are formed in appreciable amounts. Furthermore, the nitration reaction has saturation kinetics and is completely inhibited by H4B. Finally, Trp is shown to stimulate NO synthesis by deiNOS from Arg. These observations indicate that the nitration reaction occurs by Trp binding to a site that binds pterins in the mammalian enzymes. Preparation of deiNOS and TrpRS II—DeiNOS was cloned with a N-terminal His6-affinity tag, expressed in Escherichia coli BL21(DE3) cells, and purified by nickel-nitrilotriacetic acid affinity chromatography (Qiagen) as described previously (14Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar, 31Buddha M.R. Keery K. Crane B.R. Proc. Natl. Acad. Sci. 2004; (U. S. A., in press)PubMed Google Scholar). Notably, to improve solubility, deiNOS was eluted from the nickel column with 25 mm HEPES (7.5), 500 mm NaCl, 200 mm imdazole, 0.25 m sucrose and then rapidly washed 10 times in a centriprep concentrator with a solubilizing buffer (SB) made of 50 mm Tris (pH 7.5), 200 mm NaCl, 0.3 m l -arginine (Arg), 20 mm l -tryptophan. Under these conditions the protein could be concentrated to 40 mg/ml. TrpRS II was expressed, purified, and concentrated up to 40 mg/ml (31Buddha M.R. Keery K. Crane B.R. Proc. Natl. Acad. Sci. 2004; (U. S. A., in press)PubMed Google Scholar). To obtain a 1:1 complex with deiNOS, 100 μl of deiNOS (1 mm in SB) was mixed with 100 μl of TrpRS II (1 mm in 50 mm Tris (pH 7.5), 150 mm NaCl). Preparation of iNOS Reductase—The iNOS reductase construct was prepared from a plasmid containing cDNA for full-length iNOS using the polymerase chain reaction (PCR). Primers were made to amplify the final 621 residues of human iNOS-2A type 1, starting immediately following the calmodulin binding domain (residue 533). The PCR product was cloned into pET28 (Novagen) and expresed in E. coli BL21(DE3) cells. NOSred was purified using nickel-nitrilotriacetic acid metal-affinity chromatography under standard procedures. Synthesis of 4-Nitro-Trp—4-Nitro-dl -tryptophan was synthesized from commercial 4-nitroindole by conversion of the latter to 4-nitrogramine using the procedure of Melhado and Brodsky (19Melhado L.L. Brodsky J.L. J. Org. Chem. 1988; 53: 3852-3855Crossref Scopus (16) Google Scholar). The 4-nitrogramine was then converted to the hydrochloride salt of 4-nitro-dl -tryptophan in three steps by modification of the procedure of Settimo (20Da Settimo A. Ann. Chim. (Rome). 1962; 52: 17-24Google Scholar). The free amino acid was obtained from the hydrochloride salt by cation-exchange chromatography. DeiNOS Nitration from Hydrogen Peroxide—50 μm deiNOS in SB was treated with 20 mm hydrogen peroxide, and the reaction mix was monitored for 2 h at 22 °C. Similarly, 50 μm complex prepared as mentioned above was treated with 20 mm hydrogen peroxide and incubated at 22 °C for 2 h. Products of the reaction were analyzed by reverse-phase HPLC (see below) and progress of the reaction was followed by the increase in absorbance at 400 nm. Due to the relatively small extinction coefficient of nitro-Trp, ∼10–50 times more deiNOS was used for nitration reactions that is typical for standard NOS assays. Nitro-Trp formation as a function of Trp concentration was carried out with peroxide, deiNOS, and the TrpRS II-deiNOS complex exchanged in SB that did not contain tryptophan; final reaction mixtures were made up in buffers containing 50 mm Tris (pH 7.5), 200 mm NaCl, 0.3 m Arg, and increasing concentrations of tryptophan (1–20 mm). In the absence of sucrose/imidazole, deiNOS requires a high concentration of Arg for solubility. Progress of the nitration reaction was monitored by following the increase in absorbance at 400 nm and applying an extinction coefficient for nitro-Trp of ϵ400 = 3000 m–1 cm–1: ϵ400 = 4200 m–1 cm–1 was used to follow nitro-Tyr production (4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar). Because of the relatively large concentrations of deiNOS employed in the reactions, 0.2-cm cells were used to maintain an optical density under 1.0 during kinetic assays. Nitration Reaction from Mammalian NOS Reductase—For nitration reactions using mammalian reductase (mNOSred) instead of peroxide, 50 μm deiNOS or deiNOS-TrpRS II complex was treated with 10 μm iNOS reductase, 2 μm FAD, 20 μm NADPH, in presence or absence of 5–50 μm H4B (Km of H4B for deiNOS = 10 μm (14Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar)) and in the presence and absence of 30 μm ATP. Non-enzymatic Nitration of Tryptophan—Hydrogen peroxide and nitrite were used to generate nitrating agents, which primarily include peroxynitrite (4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar, 21Alvarez B. Rubbo H. Kirk M. Barnes S. Freeman B.A. Radi R. Chem. Res. Toxicol. 1996; 9: 390-396Crossref PubMed Scopus (232) Google Scholar). Hydrogen peroxide (10 mm in 0.1 m potassium phosphate buffer (pH 6.9)) was treated with sodium nitrite (10 mm in 0.1 m potassium phosphate buffer (pH 6.9)) and vortexed for about 1 min. The vortexed mixture was added to l -tryptophan (10 mm), and the reaction mixture was incubated at 37 °C for about 1 h, and the products were evaluated using reverse-phase HPLC, mass spectroscopy, and UV-visible spectroscopy. Purification of Nitration Products—The reaction mixture was passed through Supelco's Bio Wide Pore C18-5 column (25 cm × 4 mm) with pore size of 300 Å. A gradient was run using buffers A (0.07% trifluoroacetic acid in water) and B (0.07% trifluoroacetic acid in acetonitrile) and the products collected, lyophilized, and analyzed using Micromass ZMD 4000 mass spectrometer operated in the positive ion mode with a capillary voltage of 3000 V and cone voltage of 3.0 V. NOS Assays—Nitrite formation by deiNOS was monitored using the Griess reagents, whereas NO production was followed with the oxyhemoglobin assay (14Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar, 22Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). Production of 4-Nitro-tryptophan by deiNOS-TrpRS II—D. radiodurans NOS catalyzes the production of nitro-tryptophan and nitrite/nitrate from Arg or NHA in the absence or presence of TrpRS II (Table I). Nitrite is an end product of NO or related species (e.g. nitroxyl, nitrosium, collectively hereafter referred to as [NO]) reacting in oxygenated solution and a typical marker of NOS activity. As has been demonstrated for mNOS under some conditions (22Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), deiNOS will catalyze nitrite production from Arg and peroxide (Table I). Alternatively, we employed a surrogate mammalian NOSred and NADPH to drive the reaction in the absence of peroxide (14Adak S. Bilwes A.M. Panda K. Hosfield D. Aulak K.S. McDonald J.F. Tainer J.A. Getzoff E.D. Crane B.R. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 107-112Crossref PubMed Scopus (124) Google Scholar) (Table I). Human inducible NOSred was chosen as the best electron donor after also testing rat neuronal NOSred and four other homologous multiflavin reductases from B. subtilis and E. coli. Products were analyzed by HPLC (Fig. 1A), mass-spectroscopy (Fig. 1B), and optical spectroscopy (Fig. 1B). Under all conditions tested, the reaction produced one nitro-Trp isomer, which elutes on a reverse-phase HPLC column at the same retention time as a synthetic 4-nitro-Trp standard (Fig. 1A). The product was further confirmed to be 4-nitro-Trp by its characteristic absorption spectrum (Fig. 1B) and mass spectrum (Fig. 1B), whose parent ion and fragments also match the 4-nitro-Trp standard. Consistent with previous reports of nonspecific Trp nitration (4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar, 5Herold S. Shivashankar K. Mehl M. Biochemistry. 2002; 41: 13460-13472Crossref PubMed Scopus (62) Google Scholar, 6Herold S. Free Radic. Biol. Med. 2004; 36: 565-579Crossref PubMed Scopus (53) Google Scholar, 21Alvarez B. Rubbo H. Kirk M. Barnes S. Freeman B.A. Radi R. Chem. Res. Toxicol. 1996; 9: 390-396Crossref PubMed Scopus (232) Google Scholar) reactions of Trp with nitrite and peroxide (a peroxynitrite generating system (4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar, 21Alvarez B. Rubbo H. Kirk M. Barnes S. Freeman B.A. Radi R. Chem. Res. Toxicol. 1996; 9: 390-396Crossref PubMed Scopus (232) Google Scholar)) produced primarily a product whose much longer HPLC retention time and mass spectrum identify it as N1-nitro-Trp (data not shown) and a minor product (Fig. 1A) with a mass and elution profile consistent with the more favorable 6-nitro-Trp isomer (4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar, 5Herold S. Shivashankar K. Mehl M. Biochemistry. 2002; 41: 13460-13472Crossref PubMed Scopus (62) Google Scholar). Comparisons between HPLC elution areas of the 4-nitro-Trp product and absorption changes in the reaction mixture indicated that 4-nitro-Trp formation accounted for at least 80% of the absorbance change at 400 nm (ΔA400). No other small molecule products with far UV-visible absorption were observed in appreciable quantities. Thus, kinetics of Trp nitration were followed by monitoring (ΔA400). Addition of 50 units/ml superoxide dismutase also did not significantly reduce the production of nitro-Trp by deiNOS in either the reaction with peroxide or with mNOSred.Table IFormation of nitro-tryptophan and nitrite by deiNOS under various conditionsReaction monitoredaUnless indicated, all reactions were carried out in the presence of 20 mm Trp at 22 °C.kcat (4-nitro-tryptophan)kcat (nitrite)×100 min-1×100 min-1deiNOS + mRedbMammalian human inducible NOS reductase domain and NADPH.1.5 ± 0.152.3 ± 0.5deiNOS + TrpRS II + mRed - TrpNDcNot detectable.0.5 ± 0.2deiNOS + TrpRS II + mRed + H4Bd50 μm H4B. - TrpND4.5 ± 0.5deiNOS + TrpRS II + mRed3.2 ± 0.55.0 ± 0.5deiNOS + mRed + H4Bd50 μm H4B.ND2.5 ± 0.7deiNOS + TrpRS II + 5 mM H4B + mRed2.5 ± 0.24.8 ± 0.9deiNOS + TrpRS II + 25 mM H4B + mRed0.3 ± 0.014.3 ± 0.5deiNOS + TrpRS II + 50 mM H4B + mRedND4.9 ± 0.6deiNOS + TrpRS II + ATP + mRed5.1 ± 0.42.3 ± 0.7deiNOS + H2O23.5 ± 0.87.5 ± 0.5deiNOS + TrpRS II + H2O211.0 ± 3.015.0 ± 5.0deiNOS + ATP + H2O22.0 ± 0.87.1 ± 0.6deiNOS + TrpRS II + ATP + H2O225.0 ± 7.06.9 ± 0.5deiNOS + H2O2 + l-cysteine2.9 ± 0.67.0 ± 0.6a Unless indicated, all reactions were carried out in the presence of 20 mm Trp at 22 °C.b Mammalian human inducible NOS reductase domain and NADPH.c Not detectable.d 50 μm H4B. Open table in a new tab Stimulation of 4-Nitro Formation by TrpRS II and ATP— Addition of TrpRS II stimulates the production of 4-nitro-Trp by a factor of 2–3 whether peroxide or NOSred/NADPH drives the reaction (Table I). Surprisingly, the peroxide-catalyzed Trp nitration reaction is enhanced further by the addition of ATP (Table I). Consistent with greater incorporation of [NO] into the 4-nitro-Trp product, the amount of nitrite produced decreases as the amount of 4-nitro-Trp increases when ATP is present. ATP has no effect on 4-nitro-Trp or [NO] formation by deiNOS alone. ATP binds tightly to TrpRS II (31Buddha M.R. Keery K. Crane B.R. Proc. Natl. Acad. Sci. 2004; (U. S. A., in press)PubMed Google Scholar) and is required for Trp-adenylation by the synthetase. The Effects of Pterin on 4-Nitro-Trp and NO Production—Trp acts analogously to H4B in the deiNOS catalyzed nitration reaction. Similar to mNOS (22Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar), production of [NO] from Arg and mNOSred by deiNOS is enhanced ∼10× by H4B (Table I). This is consistent with the role of H4B in donating an electron to the NOS heme center during oxygen activation (8Stuehr D.J. Santolini J. Wang Z. Wei C. Adak S. J. Biol. Chem. 2004; 279: 36167-36170Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 18Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (166) Google Scholar). Surprisingly, in the absence of H4B, but presence of Trp, nitrite is still produced from Arg at nearly equal amounts (despite 4-nitro-Trp also being formed) (Table I). Notably, in the presence of Trp, H4B allows nitrite production but inhibits 4-nitro-Trp formation with increasing concentration until no 4-nitro-Trp is formed at an H4B:deiNOS-TrpRS II stoichiometry of 1:1 (Table I). H4B does not reduce the amount of nitrated product (A400) in the non-enzymatic nitrite/peroxide reaction nor does it affect nitrite formation by deiNOS in the presence of Trp. Thus, the ability of H4B to inhibit nitro-Trp formation but not nitrite formation suggests that Trp binds in the deiNOS pterin site and furthermore can stimulate oxidation of Arg from this position. We were unable to detect NO production from deiNOS-TrpRS II in the presence of Trp by the oxy-hemoglobin assay. NO production from Trp is of interest because H4B-depleted mNOS will still produce nitrite in oxygenated solution but does so through nitroxyl (NO–), rather than NO formation (22Adak S. Wang Q. Stuehr D.J. J. Biol. Chem. 2000; 275: 33554-33561Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar, 23Rusche K.M. Spiering M.M. Marletta M.A. Biochemistry. 1998; 37: 15503-15512Crossref PubMed Scopus (168) Google Scholar). Trp Nitration by deiNOS Exhibits Saturation Behavior—The dependence of 4-nitro-Trp formation by deiNOS on Trp concentration exhibits saturation behavior with an observed Michaelis constant (Km) of 2.8 ± 0.6 mm (Fig. 2). Addition of TrpRS II increases the effective Vmax two to three times and decreases Km to 1.5 ± 0.5 mm (Fig. 2). Such saturation kinetics also indicate that 4-nitro-Trp production involves Trp binding to deiNOS. A different concentration dependence would be expected for reaction of free Trp with a diffusing RNS generated by deiNOS. We also tested inhibition of Trp nitration by the peroxynitrite scavanger cysteine (21Alvarez B. Rubbo H. Kirk M. Barnes S. Freeman B.A. Radi R. Chem. Res. Toxicol. 1996; 9: 390-396Crossref PubMed Scopus (232) Google Scholar). Although 1 mm cysteine completely prevented nitro-Trp formation from nitrite and peroxide, 4-nitro-Trp formation by deiNOS decreased only slightly (15%) in the presence of cysteine (Table I). Tyrosine, the more reactive amino acid for nitration (24Bonnet R. Nicolaidou P. Heterocycles. 1977; 7: 637-659Crossref Google Scholar), is also nitrated by dei-NOS but with lower turnover numbers than Trp. At the Tyr concentrations we could evaluate, the reaction did not saturate and appeared to be roughly first order (Fig. 2). The regioselective nitration reaction catalyzed by deiNOS that produces only 4-nitro-Trp is in marked contrast to non-enzymatic and heme protein catalyzed Trp nitration reactions. Agents capable of nitrating aromatic aminoacids include peroxynitrite, nitrous acid, nitrite and hydrogen peroxide, nitric oxide and dioxygen, and acyl nitrates (4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar, 11Koppenol W.H. Free Radic. Biol. Med. 1998; 25: 385-391Crossref PubMed Scopus (303) Google Scholar, 12Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Crossref PubMed Scopus (242) Google Scholar, 24Bonnet R. Nicolaidou P. Heterocycles. 1977; 7: 637-659Crossref Google Scholar). Moreover, peroxidases in the presence of nitrite and H2O2, and myoglobin in the presence of nitrite, NO, or O2, are well characterized in their ability to nitrate Trp (4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar, 5Herold S. Shivashankar K. Mehl M. Biochemistry. 2002; 41: 13460-13472Crossref PubMed Scopus (62) Google Scholar, 6Herold S. Free Radic. Biol. Med. 2004; 36: 565-579Crossref PubMed Scopus (53) Google Scholar, 21Alvarez B. Rubbo H. Kirk M. Barnes S. Freeman B.A. Radi R. Chem. Res. Toxicol. 1996; 9: 390-396Crossref PubMed Scopus (232) Google Scholar, 25Kato Y. Kawakishi S. Aoki T. Itakura K. Osawa T. Biochem. Biophys. Res. Commun. 1997; 234: 82-84Crossref PubMed Scopus (57) Google Scholar, 26Padmaya S. Ramazenian M.S. Bonds P.L. Koppenol W.H. Redox Rep. 1996; 2: 173-177Crossref PubMed Scopus (59) Google Scholar). These reactions generate a large number of products which include the 4-nitro, 5-nitro, 6-nitro, 7-nitro, N1-nitro, and N1-nitroso derivatives of Trp as well as hydroxylated Trp and N-formylkinureine (4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar, 5Herold S. Shivashankar K. Mehl M. Biochemistry. 2002; 41: 13460-13472Crossref PubMed Scopus (62) Google Scholar, 6Herold S. Free Radic. Biol. Med. 2004; 36: 565-579Crossref PubMed Scopus (53) Google Scholar, 21Alvarez B. Rubbo H. Kirk M. Barnes S. Freeman B.A. Radi R. Chem. Res. Toxicol. 1996; 9: 390-396Crossref PubMed Scopus (232) Google Scholar, 25Kato Y. Kawakishi S. Aoki T. Itakura K. Osawa T. Biochem. Biophys. Res. Commun. 1997; 234: 82-84Crossref PubMed Scopus (57) Google Scholar, 26Padmaya S. Ramazenian M.S. Bonds P.L. Koppenol W.H. Redox Rep. 1996; 2: 173-177Crossref PubMed Scopus (59) Google Scholar). Trp nitration reactions catalyzed by hemeproteins involve multiple competing reaction pathways; that can involve peroxynitrite intermediates and indole radicals generated by compound I or II peroxidase species (4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar, 11Koppenol W.H. Free Radic. Biol. Med. 1998; 25: 385-391Crossref PubMed Scopus (303) Google Scholar, 12Hughes M.N. Biochim. Biophys. Acta. 1999; 1411: 263-272Crossref PubMed Scopus (242) Google Scholar, 24Bonnet R. Nicolaidou P. Heterocycles. 1977; 7: 637-659Crossref Google Scholar). Given the nonspecific character of these reactions, it is then remarkable that deiNOS produces a single nitrated derivative of Trp, the same 4-nitro isomer produced by the Streptomyces NOS in the thaxtomin phytotoxin. deiNOS likely interacts with Trp to protect the more reactive sites on the indole ring from modification. The mammalian NOS cofactor H4B does not significantly affect [NO] production by deiNOS in the peroxide assay but completely inhibits production of nitro-Trp. This result indicates that Trp nitration by deiNOS does not occur by reaction between free Trp and a diffusible nitrating agent derived from deiNOS and Arg. Furthermore, the inability of the peroxynitrite scavanger cysteine to prevent nitro-Trp formation rules out the obligatory participation of free preoxynitrite. Saturation behavior of the nitration reaction with Trp concentration also supports Trp binding to deiNOS. Neither saturation kinetics nor comparably high product yields are observed with Tyr, which is at least as reactive toward nitrating agents as Trp (24Bonnet R. Nicolaidou P. Heterocycles. 1977; 7: 637-659Crossref Google Scholar). Inhibition of 4-nitro-Trp formation by H4B indicates that rates saturate with Trp concentration because Trp binds in the pterin site. In Bacillus subtilis NOS, H4B accelerates decay of the heme ferrous-oxy species by presumably acting as a rapid electron donor to heme, akin to its role in mammalian NOS catalysis (8Stuehr D.J. Santolini J. Wang Z. Wei C. Adak S. J. Biol. Chem. 2004; 279: 36167-36170Abstract Full Text Full Text PDF PubMed Scopus (412) Google Scholar, 17Adak S. Aulak K. Stuehr D.J. J. Biol. Chem. 2002; 277: 16167-16171Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar, 18Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (166) Google Scholar, 27Sorlie M. Gorren A.C.F. Marchal S. Shimizu T. Lange R. Andersson K.K. Mayer B. J. Biol. Chem. 2003; 278: 48602-48610Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). The dependence of deiNOS on reduced pterins (15Pant K. Bilwes A.M. Adak S. Stuehr D.J. Crane B.R. Biochemistry. 2002; 41: 11071-11079Crossref PubMed Scopus (122) Google Scholar) or Trp for [NO] formation from Arg could imply that deiNOS maintains this mechanism but that Trp can substitute for H4B as an electron donor. This is surprising, given that the reduction potential of the Trp radical (1.0 V (28Tommos C. Skalicky J.J. Pilloud D.L. Wand A.J. Dutton P.L. Biochemistry. 1999; 38: 9495-9507Crossref PubMed Scopus (177) Google Scholar)) is expected to be much higher than that for H3B+ (0.15–0.3 V (Ref. 29Gorren A.C.F. Kungl A.J. Schmidt K. Werner E.R. Mayer B. Nitric Oxide-Biol. Chem. 2001; 5: 176-186Crossref PubMed Scopus (61) Google Scholar and references therein)). Nonetheless, the protein and or protonation environment could modulate these potentials (27Sorlie M. Gorren A.C.F. Marchal S. Shimizu T. Lange R. Andersson K.K. Mayer B. J. Biol. Chem. 2003; 278: 48602-48610Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), and indeed, NOSs do provide a negative electrostatic field at the pterin site that will facilitate oxidation of cofactors (18Wei C.C. Crane B.R. Stuehr D.J. Chem. Rev. 2003; 103: 2365-2383Crossref PubMed Scopus (166) Google Scholar). Alternatively, Trp could be oxidized by the compound I-like oxidation state of NOS, as is seen in reactions by peroxidases (4Sala A. Stefania N. Roncone R. Casella L. Monzani E. Eur. J. Biochem. 2004; 271: 2841-2851Crossref PubMed Scopus (41) Google Scholar). But such a mechanism does not explain how Trp stimulates nitrite production from deiNOS-NOSred. Coupling oxidation of Trp to the heme chemistry that oxidizes Arg to NO is an attractive means of providing a reactive indole radical to the [NO] species derived at the heme site. Such a mechanism would limit the toxic effects of [NO] diffusion away from deiNOS. If [NO] coupling with the radical proceeded prior to [NO] reaction with oxygen, a 4-nitroso-Trp would be a likely initial product (see Supplemental Fig. 3). As C-nitroso-Trp may be unstable to both oxidation and nitroso migration (24Bonnet R. Nicolaidou P. Heterocycles. 1977; 7: 637-659Crossref Google Scholar, 30Castro A. Iglesias E. Leis J.R. Pena M.E. Tato J.V. Williams D.L.H. J. Chem. Soc. Perkin Trans. II. 1986; 2: 1165-1168Crossref Scopus (33) Google Scholar), it is not surprising no such species was observed. Significantly, TrpRS II activation of deiNOS catalyzed nitration is greater in the presence of ATP. TrpRS II requires ATP to adenylate Trp prior to tRNA charging. deiNOS itself does not interact with ATP nor does ATP affect nitro-Trp or [NO] production of deiNOS alone. Thus, 5′-adenyl-Trp, produced by TrpRS II, may be a preferred substrate for deiNOS nitration (see supplemental Fig. 3). The 5′-adenyl-Trp is unstable to hydrolysis and difficult to isolate as an intermediate (13Doublie S. Bricogne G. Gilmore C. Carter C.W. Structure (Lond.). 1995; 3: 17-31Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar). Alternatively, ATP binding to TrpRS II may influence deiNOS in a manner that stimulates Trp nitration. The ability of TrpRS II and ATP to enhance the deiNOS nitration reaction underscores the functional coupling of these two enzymes. Many enzymes involved in secondary metaboilsm, such as non-ribosomal peptide synthases, polyketide synthases, terpene synthases, and taxadiene synthases, have relatively slow turnover numbers that often range between 0.001 and 0.8 min–1 (The Comprehensive Enzyme Information System, www.brenda.uni-koeln.de). Furthermore, enzymes that metabolize Trp, such as tryptophan transaminase, peptide-tryptophan dioxygenase, tryptophanase, and l-tryptophan oxidase, have Km values for Trp of ∼1mm. Thus, the catalytic parameters for Trp nitration by deiNOS are well within the range expected for a biosynthetic function. The production of 4-nitro-Trp isomer by both deiNOS and the Streptomyces NOS that biosynthesizes thaxtomin may not be coincidental, particularly if both reactions require Trp binding to NOS proteins that do indeed share high homology in their pterin binding sites. We thank Stuart Krasnoff for assistance with mass spectrometry and Harold Scheraga and colleagues for help with HPLC. 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