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Sample records for 4-hydroxyphenylpyruvate dioxygenase 4-hppd

  1. The Role of 4-Hydroxyphenylpyruvate Dioxygenase in Enhancement of Solid-Phase Electron Transfer by Shewanella oneidensis MR-1

    SciTech Connect

    Turick, Charles E.; Beliaev, Alex S.; Zakrajsek, Brian A.; Reardon, Catherine L.; Lowy, Daniel A.; Poppy, Tara E.; Maloney, Andrea; Ekechukwu, Amy A.

    2009-05-01

    ABSTRACT - While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane associated c-type cytochromes and electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of the tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. We determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione ([2-(2- chloro- 4- methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA, which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates at which MR-1 reduces hydrous ferric oxide were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E°') of S. oneidensis MR-1. Based on our findings, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in S. oneidensis MR-1.

  2. THE ROLE OF 4-HYDROXYPHENYLPYRUVATE DIOXYGENASE IN ENHANCEMENT OF SOLID-PHASE ELECTRON TRANSFER BY SHEWANELLA ONEIDENSIS MR-1

    SciTech Connect

    Turick, C; Amy Ekechukwu, A

    2007-06-01

    While mechanistic details of dissimilatory metal reduction are far from being understood, it is postulated that the electron transfer to solid metal oxides is mediated by outer membrane-associated c-type cytochromes and redox active electron shuttling compounds. This study focuses on the production of homogensitate in Shewanella oneidensis MR-1, an intermediate of tyrosine degradation pathway, which is a precursor of a redox cycling metabolite, pyomelanin. In this study, we determined that two enzymes involved in this pathway, 4-hydroxyphenylpyruvate dioxygenase (4HPPD) and homogentisate 1,2-dioxygenase are responsible for homogentisate production and oxidation, respectively. Inhibition of 4-HPPD activity with the specific inhibitor sulcotrione (2-(2-chloro-4-methane sulfonylbenzoyl)-1,3-cyclohexanedione), and deletion of melA, a gene encoding 4-HPPD, resulted in no pyomelanin production by S. oneidensis MR-1. Conversely, deletion of hmgA which encodes the putative homogentisate 1,2-dioxygenase, resulted in pyomelanin overproduction. The efficiency and rates, with which MR-1 reduces hydrous ferric oxide, were directly linked to the ability of mutant strains to produce pyomelanin. Electrochemical studies with whole cells demonstrated that pyomelanin substantially increases the formal potential (E{sup o}{prime}) of S. oneidensis MR-1. Based on this work, environmental production of pyomelanin likely contributes to an increased solid-phase metal reduction capacity in Shewanella oneidensis.

  3. On 4-hydroxyphenylpyruvate dioxygenase of adult frog liver.

    PubMed

    Lindstedt, S; Odelhög, B; Rundgren, M

    1982-01-01

    1. It has been reported that 4-hydroxyphenylpyruvate dioxygenase (EC 1.13.11.27) activity in the liver from Rana esculenta is present only after autolysis of trypsin digestion, which releases a heat-and acid-stable inhibitor of low molecular mass. 2. Attempts to demonstrate similar effects with the liver enzyme from adult Rana pipiens were unsuccessful. Trypsin had only an inhibitory effect on the enzyme activity in crude extracts. 3. Both untreated and trypsin-treated enzyme had a molecular mass of about 100,000 daltons as determined by gel filtration. The pI was around pH 4.6. One pH-optimum between pH 7 and 8 was observed. 4. At pH 7.5 and 37 degrees C the basal enzyme activity was 1.3 mumol/min per g of protein. It was increased six-fold by a reductant in the presence of catalase. Fe2+ (50 muM) increased the activity further 1.6-fold when the reaction was carried out in Tris-HCl buffer, but not in potassium phosphate buffer. 5. The Km for 4-hydroxyphenylpyruvate was 50 muM and the Vmax was around 10 mumol/min per g of soluble protein with reductively activated enzyme. 6. Substrate inhibition was observed above 20 muM concentrations of 4-hydroxyphenylpyruvate.

  4. Novel bacterial bioassay for a high-throughput screening of 4-hydroxyphenylpyruvate dioxygenase inhibitors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant 4-hydroxyphenylpyruvate dioxygenase (HPPD) is the molecular target of a range of synthetic ß-triketone herbicides that are currently used commercially. Their mode of action is based on an irreversible inhibition of HPPD. Therefore, this inhibitory capacity was used to develop a whole cell colo...

  5. 4-Hydroxyphenylpyruvate dioxygenase inhibitors in combination with safeners: solutions for modern and sustainable agriculture.

    PubMed

    Ahrens, Hartmut; Lange, Gudrun; Müller, Thomas; Rosinger, Chris; Willms, Lothar; van Almsick, Andreas

    2013-09-02

    Inhibitors of 4-hydroxyphenylpyruvate dioxygenase (HPPD) prevent plant carotenoid pigment formation, which in turn leads to chlorophyll degradation. This "bleaching" herbicide mode of action provides weed-control products for various crops, such as rice, corn, and cereals. Combinations with suitable safeners allow the full exploitation of the potential of this compound class to selectively control major weed problems, including rapidly increasing cases of resistance against other important herbicide classes.

  6. Synthesis and bioevaluation of pyrazole-benzimidazolone hybrids as novel human 4-Hydroxyphenylpyruvate dioxygenase inhibitors.

    PubMed

    Xu, Yu-Ling; Lin, Hong-Yan; Ruan, Xu; Yang, Sheng-Gang; Hao, Ge-Fei; Yang, Wen-Chao; Yang, Guang-Fu

    2015-03-06

    4-Hydroxyphenylpyruvate dioxygenase (HPPD), an essential enzyme in tyrosine catabolism, is an important target for treating type I tyrosinemia. Inhibition of HPPD can effectively alleviate the symptoms of type I tyrosinemia. However, only one commercial HPPD inhibitor, 2-(2-nitro-4-trifluoromethylbenzoyl) cyclohexane-1,3-dione (NTBC), has been available for clinical use so far. In the present study, a series of novel pyrazole-benzimidazolone hybrids were designed, synthesized and evaluated as potent human HPPD inhibitors. Most of the new compounds displayed significant inhibitory activity against the recombinant human HPPD. Moreover, compound 9l was identified as the most potent candidate with IC50 value of 0.021 μM against recombinant human HPPD, about 3-fold more potent than NTBC. Thus the pyrazole-benzimidazolone hybrid has great potential to be further developed for the treatment of type I tyrosinemia.

  7. Broad 4-Hydroxyphenylpyruvate Dioxygenase Inhibitor Herbicide Tolerance in Soybean with an Optimized Enzyme and Expression Cassette[W][OPEN

    PubMed Central

    Siehl, Daniel L.; Tao, Yumin; Albert, Henrik; Dong, Yuxia; Heckert, Matthew; Madrigal, Alfredo; Lincoln-Cabatu, Brishette; Lu, Jian; Fenwick, Tamara; Bermudez, Ericka; Sandoval, Marian; Horn, Caroline; Green, Jerry M.; Hale, Theresa; Pagano, Peggy; Clark, Jenna; Udranszky, Ingrid A.; Rizzo, Nancy; Bourett, Timothy; Howard, Richard J.; Johnson, David H.; Vogt, Mark; Akinsola, Goke; Castle, Linda A.

    2014-01-01

    With an optimized expression cassette consisting of the soybean (Glycine max) native promoter modified for enhanced expression driving a chimeric gene coding for the soybean native amino-terminal 86 amino acids fused to an insensitive shuffled variant of maize (Zea mays) 4-hydroxyphenylpyruvate dioxygenase (HPPD), we achieved field tolerance in transgenic soybean plants to the HPPD-inhibiting herbicides mesotrione, isoxaflutole, and tembotrione. Directed evolution of maize HPPD was accomplished by progressively incorporating amino acids from naturally occurring diversity and novel substitutions identified by saturation mutagenesis, combined at random through shuffling. Localization of heterologously expressed HPPD mimicked that of the native enzyme, which was shown to be dually targeted to chloroplasts and the cytosol. Analysis of the native soybean HPPD gene revealed two transcription start sites, leading to transcripts encoding two HPPD polypeptides. The N-terminal region of the longer encoded peptide directs proteins to the chloroplast, while the short form remains in the cytosol. In contrast, maize HPPD was found almost exclusively in chloroplasts. Evolved HPPD enzymes showed insensitivity to five inhibitor herbicides. In 2013 field trials, transgenic soybean events made with optimized promoter and HPPD variant expression cassettes were tested with three herbicides and showed tolerance to four times the labeled rates of mesotrione and isoxaflutole and two times the labeled rates of tembotrione. PMID:25192697

  8. A root-expressed L-phenylalanine:4-hydroxyphenylpyruvate aminotransferase is required for tropane alkaloid biosynthesis in Atropa belladonna.

    PubMed

    Bedewitz, Matthew A; Góngora-Castillo, Elsa; Uebler, Joseph B; Gonzales-Vigil, Eliana; Wiegert-Rininger, Krystle E; Childs, Kevin L; Hamilton, John P; Vaillancourt, Brieanne; Yeo, Yun-Soo; Chappell, Joseph; DellaPenna, Dean; Jones, A Daniel; Buell, C Robin; Barry, Cornelius S

    2014-09-01

    The tropane alkaloids, hyoscyamine and scopolamine, are medicinal compounds that are the active components of several therapeutics. Hyoscyamine and scopolamine are synthesized in the roots of specific genera of the Solanaceae in a multistep pathway that is only partially elucidated. To facilitate greater understanding of tropane alkaloid biosynthesis, a de novo transcriptome assembly was developed for Deadly Nightshade (Atropa belladonna). Littorine is a key intermediate in hyoscyamine and scopolamine biosynthesis that is produced by the condensation of tropine and phenyllactic acid. Phenyllactic acid is derived from phenylalanine via its transamination to phenylpyruvate, and mining of the transcriptome identified a phylogenetically distinct aromatic amino acid aminotransferase (ArAT), designated Ab-ArAT4, that is coexpressed with known tropane alkaloid biosynthesis genes in the roots of A. belladonna. Silencing of Ab-ArAT4 disrupted synthesis of hyoscyamine and scopolamine through reduction of phenyllactic acid levels. Recombinant Ab-ArAT4 preferentially catalyzes the first step in phenyllactic acid synthesis, the transamination of phenylalanine to phenylpyruvate. However, rather than utilizing the typical keto-acid cosubstrates, 2-oxoglutarate, pyruvate, and oxaloacetate, Ab-ArAT4 possesses strong substrate preference and highest activity with the aromatic keto-acid, 4-hydroxyphenylpyruvate. Thus, Ab-ArAT4 operates at the interface between primary and specialized metabolism, contributing to both tropane alkaloid biosynthesis and the direct conversion of phenylalanine to tyrosine.

  9. Hydroperoxylation by Hydroxyethylphosphonate Dioxygenase

    PubMed Central

    2009-01-01

    Hydroxyethylphosphonate dioxygenase (HEPD) catalyzes the O2-dependent cleavage of the carbon−carbon bond of 2-hydroxyethylphosphonate (2-HEP) to afford hydroxymethylphosphonate (HMP) and formate without input of electrons or use of any organic cofactors. Two mechanisms have been proposed to account for this reaction. One involves initial hydroxylation of substrate to an acetal intermediate and its subsequent attack onto an Fe(IV)-oxo species. The second mechanism features initial hydroperoxylation of substrate followed by a Criegee rearrangement. To distinguish between the two mechanisms, substrate analogues were synthesized and presented to the enzyme. Hydroxymethylphosphonate was converted into phosphate and formate, and 1-hydroxyethylphosphonate was converted to acetylphosphate, which is an inhibitor of the enzyme. These results provide strong support for a Criegee rearrangement with a phosphorus-based migrating group and require that the O−O bond of molecular oxygen is not cleaved prior to substrate activation. (2R)-Hydroxypropylphosphonate partitioned between conversion to 2-oxopropylphosphonate and hydroxymethylphosphonate, with the latter in turn converted to phosphate and formate. Collectively, these results support a mechanism that proceeds by hydroperoxylation followed by a Criegee rearrangement. PMID:19839620

  10. Hydroperoxylation by hydroxyethylphosphonate dioxygenase.

    PubMed

    Whitteck, John T; Cicchillo, Robert M; van der Donk, Wilfred A

    2009-11-11

    Hydroxyethylphosphonate dioxygenase (HEPD) catalyzes the O(2)-dependent cleavage of the carbon-carbon bond of 2-hydroxyethylphosphonate (2-HEP) to afford hydroxymethylphosphonate (HMP) and formate without input of electrons or use of any organic cofactors. Two mechanisms have been proposed to account for this reaction. One involves initial hydroxylation of substrate to an acetal intermediate and its subsequent attack onto an Fe(IV)-oxo species. The second mechanism features initial hydroperoxylation of substrate followed by a Criegee rearrangement. To distinguish between the two mechanisms, substrate analogues were synthesized and presented to the enzyme. Hydroxymethylphosphonate was converted into phosphate and formate, and 1-hydroxyethylphosphonate was converted to acetylphosphate, which is an inhibitor of the enzyme. These results provide strong support for a Criegee rearrangement with a phosphorus-based migrating group and require that the O-O bond of molecular oxygen is not cleaved prior to substrate activation. (2R)-Hydroxypropylphosphonate partitioned between conversion to 2-oxopropylphosphonate and hydroxymethylphosphonate, with the latter in turn converted to phosphate and formate. Collectively, these results support a mechanism that proceeds by hydroperoxylation followed by a Criegee rearrangement.

  11. Synthesis of Substituted Catechols using Nitroarene Dioxygenases

    DTIC Science & Technology

    2006-01-01

    31] Keenan BG, Leungsakul T, Smets BF, Wood TK. Saturation muta- genesis of Burkholderia cepacia R34 2,4-dinitrotoluene dioxygenase at DntAc valine...for the nitrobenzene dioxygenase from Comamonas sp. strain JS765 [19] and the 2,4-dinitotoluene dioxygenase from Burkholderia sp. strain DNT [20] and...2,4-dinitrotoluene dioxygenase (24DDO) from Burkholderia sp. strain DNT [20]. The plasmid was derived by subcloning the 6.7-kb SacI:SalI restriction

  12. The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa Is a 3-Mercaptopropionate Dioxygenase*

    PubMed Central

    Tchesnokov, Egor P.; Fellner, Matthias; Siakkou, Eleni; Kleffmann, Torsten; Martin, Lois W.; Aloi, Sekotilani; Lamont, Iain L.; Wilbanks, Sigurd M.; Jameson, Guy N. L.

    2015-01-01

    Thiol dioxygenation is the initial oxidation step that commits a thiol to important catabolic or biosynthetic pathways. The reaction is catalyzed by a family of specific non-heme mononuclear iron proteins each of which is reported to react efficiently with only one substrate. This family of enzymes includes cysteine dioxygenase, cysteamine dioxygenase, mercaptosuccinate dioxygenase, and 3-mercaptopropionate dioxygenase. Using sequence alignment to infer cysteine dioxygenase activity, a cysteine dioxygenase homologue from Pseudomonas aeruginosa (p3MDO) has been identified. Mass spectrometry of P. aeruginosa under standard growth conditions showed that p3MDO is expressed in low levels, suggesting that this metabolic pathway is available to the organism. Purified recombinant p3MDO is able to oxidize both cysteine and 3-mercaptopropionic acid in vitro, with a marked preference for 3-mercaptopropionic acid. We therefore describe this enzyme as a 3-mercaptopropionate dioxygenase. Mössbauer spectroscopy suggests that substrate binding to the ferrous iron is through the thiol but indicates that each substrate could adopt different coordination geometries. Crystallographic comparison with mammalian cysteine dioxygenase shows that the overall active site geometry is conserved but suggests that the different substrate specificity can be related to replacement of an arginine by a glutamine in the active site. PMID:26272617

  13. 2,4-Dinitrotoluene dioxygenase from Burkholderia sp. strain DNT: similarity to naphthalene dioxygenase.

    PubMed Central

    Suen, W C; Haigler, B E; Spain, J C

    1996-01-01

    2,4-Dinitrotoluene (DNT) dioxygenase from Burkholderia sp. strain DNT catalyzes the initial oxidation of DNT to form 4-methyl-5-nitrocatechol (MNC) and nitrite. The displacement of the aromatic nitro group by dioxygenases has only recently been described, and nothing is known about the evolutionary origin of the enzyme systems that catalyze these reactions. We have shown previously that the gene encoding DNT dioxygenase is localized on a degradative plasmid within a 6.8-kb NsiI DNA fragment (W.-C. Suen and J. C. Spain, J. Bacteriol. 175:1831-1837, 1993). We describe here the sequence analysis and the substrate range of the enzyme system encoded by this fragment. Five open reading frames were identified, four of which have a high degree of similarity (59 to 78% identity) to the components of naphthalene dioxygenase (NDO) from Pseudomonas strains. The conserved amino acid residues within NDO that are involved in cofactor binding were also identified in the gene encoding DNT dioxygenase. An Escherichia coli clone that expressed DNT dioxygenase converted DNT to MNC and also converted naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. In contrast, the E. coli clone that expressed NDO did not oxidize DNT. Furthermore, the enzyme systems exhibit similar broad substrate specificities and can oxidize such compounds as indole, indan, indene, phenetole, and acenaphthene. These results suggest that DNT dioxygenase and the NDO enzyme system share a common ancestor. PMID:8759857

  14. Spectroscopic Studies of the Catechol Dioxygenases.

    ERIC Educational Resources Information Center

    Que, Lawrence Jr.

    1985-01-01

    The catechol dioxygenases are bacterial iron-containing enzymes that catalyze the oxidative cleavage of catechols. These enzymes serve as a component of nature's mechanisms for degrading aromatic compounds in the environment. The structure and mechanistic aspects of these enzymes are described. (JN)

  15. Discovery and characterization of a second mammalian thiol dioxygenase, cysteamine dioxygenase.

    PubMed

    Dominy, John E; Simmons, Chad R; Hirschberger, Lawrence L; Hwang, Jesse; Coloso, Relicardo M; Stipanuk, Martha H

    2007-08-31

    There are only two known thiol dioxygenase activities in mammals, and they are ascribed to the enzymes cysteine dioxygenase (CDO) and cysteamine (2-aminoethanethiol) dioxygenase (ADO). Although many studies have been dedicated to CDO, resulting in the identification of its gene and even characterization of the tertiary structure of the protein, relatively little is known about cysteamine dioxygenase. The failure to identify the gene for this protein has significantly hampered our understanding of the metabolism of cysteamine, a product of the constitutive degradation of coenzyme A, and the synthesis of taurine, the final product of cysteamine oxidation and the second most abundant amino acid in mammalian tissues. In this study we identified a hypothetical murine protein homolog of CDO (hereafter called ADO) that is encoded by the gene Gm237 and belongs to the DUF1637 protein family. When expressed as a recombinant protein, ADO exhibited significant cysteamine dioxygenase activity in vitro. The reaction was highly specific for cysteamine; cysteine was not oxidized by the enzyme, and structurally related compounds were not competitive inhibitors of the reaction. When overexpressed in HepG2/C3A cells, ADO increased the production of hypotaurine from cysteamine. Similarly, when endogenous expression of the human ADO ortholog C10orf22 in HepG2/C3A cells was reduced by RNA-mediated interference, hypotaurine production decreased. Western blots of murine tissues with an antibody developed against ADO showed that the protein is ubiquitously expressed with the highest levels in brain, heart, and skeletal muscle. Overall, these data suggest that ADO is responsible for endogenous cysteamine dioxygenase activity.

  16. Thiol Dioxygenases: Unique Families of Cupin Proteins

    PubMed Central

    Simmons, C. R.; Karplus, P. A.; Dominy, J. E.

    2011-01-01

    Proteins in the cupin superfamily have a wide range of biological functions in archaea, bacteria and eukaryotes. Although proteins in the cupin superfamily show very low overall sequence similarity, they all contain two short but partially conserved cupin sequence motifs separated by a less conserved intermotif region that varies both in length and amino acid sequence. Furthermore, these proteins all share a common architecture described as a 6-stranded β-barrel core, and this canonical cupin or “jelly roll” β-barrel is formed with cupin motif 1, the intermotif region, and cupin motif 2 each forming two of the core six β-strands in the folded protein structure. The recently obtained crystal structures of cysteine dioxygenase (CDO), with contains conserved cupin motifs, show that it has the predicted canonical cupin β-barrel fold. Although there had been no reports of CDO activity in prokaryotes, we identified a number of bacterial cupin proteins of unknown function that share low similarity with mammalian CDO and that conserve many residues in the active site pocket of CDO. Putative bacterial CDOs predicted to have CDO activity were shown to have similar substrate specificity and kinetic parameters as eukaryotic CDOs. Information gleaned from crystal structures of mammalian CDO along with sequence information for homologs shown to have CDO activity facilitated the identification of a CDO family fingerprint motif. One key feature of the CDO fingerprint motif is that the canonical metal-binding glutamate residue in cupin motif 1 is replaced by a cysteine (in mammalian CDOs) or by a glycine (bacterial CDOs). The recent report that some putative bacterial CDO homologs are actually 3-mercaptopropionate dioxygenases suggests that the CDO family may include proteins with specificities for other thiol substrates. A paralog of CDO in mammals was also identified and shown to be the other mammalian thiol dioxygenase, cysteamine dioxygenase (ADO). A tentative

  17. Autoxidation-product-initiated dioxygenases: vanadium-based, record catalytic lifetime catechol dioxygenase catalysis.

    PubMed

    Yin, Cindy-Xing; Sasaki, Yoh; Finke, Richard G

    2005-11-14

    In recent work, it was shown that V-containing polyoxometalates such as (n-Bu4N)7SiW9V3O40 or (n-Bu4N)9P2W15V3O62, as well as eight other V-containing precatalysts tested, evolve to a high activity, long catalytic lifetime (> or = 30,000-100,000 total turnovers) 3,5-di-tert-butylcatechol dioxygenase, in which Pierpont's complex [VO(DBSQ)(DTBC)]2 (where DBSQ is 3,5-di-tert-butylsemiquinone and DTBC is the 3,5-di-tert-butylcatecholate dianion) was identified as a common catalyst or catalyst resting state (Yin, C.-X.; Finke, R. G. Vanadium-Based, Extended Catalytic Lifetime Catechol Dioxygenases: Evidence For a Common Catalyst. J. Am. Chem. Soc. 2005, 127 (25), 9003-9013). Herein, those findings are followed up by studies aimed at answering the following questions about this record catalytic lifetime 3,5-di-tert-butylcatechol dioxygenase catalyst: (i) What is the key to how V leaches from, for example, seemingly robust V-containing polyoxometalate precatalysts? (ii) What is the key to the sigmoidal, apparently autocatalytic kinetics observed? (iii) What can be learned about the underlying reactions that form [VO(DBSQ)(DTBC)]2? (iv) Finally, do the answers to (i-iii) lead to any broader insights or concepts? Key findings from the present work include the fact that the reaction involves a novel, autoxidation-product-induced dioxygenase, that is, one in which the undesired autoxidation of the 3,5-di-tert-butylcatechol substrate to the corresponding benzoquinone and H2O2 turns on the desired dioxygenase catalysis via a V-leaching process which eventually yields Pierpont's complex, [VO(DBSQ)(DTBC)]2. Plausible reactions en route to [VO(DBSQ)(DTBC)]2 consistent with the kinetic data, the role of H2O2, and the relevant literature are provided. The results provide a prototype example of the little observed but likely more general concept of an autoxidation-product-initiated reaction. The results also provide considerable simplification of, and insight into, the previously

  18. The "Gln-Type" Thiol Dioxygenase from Azotobacter vinelandii is a 3-Mercaptopropionic Acid Dioxygenase.

    PubMed

    Pierce, Brad S; Subedi, Bishnu P; Sardar, Sinjinee; Crowell, Joshua K

    2015-12-29

    Cysteine dioxygenase (CDO) is a non-heme iron enzyme that catalyzes the O2-dependent oxidation of l-cysteine to produce cysteinesulfinic acid. Bacterial CDOs have been subdivided as either "Arg-type" or "Gln-type" on the basis of the identity of conserved active site residues. To date, "Gln-type" enzymes remain largely uncharacterized. It was recently noted that the "Gln-type" enzymes are more homologous with another thiol dioxygenase [3-mercaptopropionate dioxygenase (MDO)] identified in Variovorax paradoxus, suggesting that enzymes of the "Gln-type" subclass are in fact MDOs. In this work, a putative "Gln-type" thiol dioxygenase from Azotobacter vinelandii (Av) was purified to homogeneity and characterized. Steady-state assays were performed using three substrates [3-mercaptopropionic acid (3mpa), l-cysteine (cys), and cysteamine (ca)]. Despite comparable maximal velocities, the "Gln-type" Av enzyme exhibited a specificity for 3mpa (kcat/KM = 72000 M(-1) s(-1)) nearly 2 orders of magnitude greater than those for cys (110 M(-1) s(-1)) and ca (11 M(-1) s(-1)). Supporting X-band electron paramagnetic resonance (EPR) studies were performed using nitric oxide (NO) as a surrogate for O2 binding to confirm obligate-ordered addition of substrate prior to NO. Stoichimetric addition of NO to solutions of 3mpa-bound enzyme quantitatively yields an iron-nitrosyl species (Av ES-NO) with EPR features consistent with a mononuclear (S = (3)/2) {FeNO}(7) site. Conversely, two distinct substrate-bound conformations were observed in Av ES-NO samples prepared with cys and ca, suggesting heterogeneous binding within the enzymatic active site. Analytical EPR simulations are provided to establish the relative binding affinity for each substrate (3map > cys > ca). Both kinetic and spectroscopic results presented here are consistent with 3mpa being the preferred substrate for this enzyme.

  19. Hydrolase-like properties of a cofactor-independent dioxygenase.

    PubMed

    Thierbach, Sven; Büldt-Karentzopoulos, Klaudia; Dreiling, Alena; Hennecke, Ulrich; König, Simone; Fetzner, Susanne

    2012-05-29

    Mechanistic promiscuity: The (2-alkyl)-3-hydroxy-4(1H)-quinolone-cleaving dioxygenase Hod has an α/β-hydrolase fold and a Ser/His/Asp triad in its active site. Isatoic anhydride, a suicide substrate of serine hydrolases, inactivates Hod by covalent modification of the active-site serine, thus indicating that the α/β-hydrolase fold can accommodate dioxygenase chemistry without completely abandoning hydrolase-like properties.

  20. 3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Klebsiella pneumoniae, a Mg(2+)-containing dioxygenase involved in aromatic catabolism.

    PubMed Central

    Gibello, A; Ferrer, E; Martín, M; Garrido-Pertierra, A

    1994-01-01

    3,4-Dihydroxyphenylacetate 2,3-dioxygenase, an extradiol-ring-cleavage dioxygenase, has been purified from Klebsiella pneumoniae to homogeneity. The enzyme has an M(r) of 102,000 in its tetrameric form with an M(r) of 25,500 for each subunit. Unlike most other dioxygenases, the enzyme reported here contains Mg2+, as determined by atomic-absorption spectrophotometry and plasma emission metal analysis. The enzyme was shown to contain approx. 1 g-atom of Mg2+/mol of protein and we suggest an alpha 4 Mg2+ quaternary structure. This is the first report of a dioxygenase containing Mg2+ in its structure. Images Figure 1 PMID:8037662

  1. Hemoglobin: A Nitric-Oxide Dioxygenase

    PubMed Central

    Gardner, Paul R.

    2012-01-01

    Members of the hemoglobin superfamily efficiently catalyze nitric-oxide dioxygenation, and when paired with native electron donors, function as NO dioxygenases (NODs). Indeed, the NOD function has emerged as a more common and ancient function than the well-known role in O2 transport-storage. Novel hemoglobins possessing a NOD function continue to be discovered in diverse life forms. Unique hemoglobin structures evolved, in part, for catalysis with different electron donors. The mechanism of NOD catalysis by representative single domain hemoglobins and multidomain flavohemoglobin occurs through a multistep mechanism involving O2 migration to the heme pocket, O2 binding-reduction, NO migration, radical-radical coupling, O-atom rearrangement, nitrate release, and heme iron re-reduction. Unraveling the physiological functions of multiple NODs with varying expression in organisms and the complexity of NO as both a poison and signaling molecule remain grand challenges for the NO field. NOD knockout organisms and cells expressing recombinant NODs are helping to advance our understanding of NO actions in microbial infection, plant senescence, cancer, mitochondrial function, iron metabolism, and tissue O2 homeostasis. NOD inhibitors are being pursued for therapeutic applications as antibiotics and antitumor agents. Transgenic NOD-expressing plants, fish, algae, and microbes are being developed for agriculture, aquaculture, and industry. PMID:24278729

  2. Regiospecific and stereoselective hydroxylation of 1-indanone and 2-indanone by naphthalene dioxygenase and toluene dioxygenase.

    PubMed Central

    Resnick, S M; Torok, D S; Lee, K; Brand, J M; Gibson, D T

    1994-01-01

    The biotransformation of 1-indanone and 2-indanone to hydroxyindanones was examined with bacterial strains expressing naphthalene dioxygenase (NDO) and toluene dioxygenase (TDO) as well as with purified enzyme components. Pseudomonas sp. strain 9816/11 cells, expressing NDO, oxidized 1-indanone to a mixture of 3-hydroxy-1-indanone (91%) and 2-hydroxy-1-indanone (9%). The (R)-3-hydroxy-1-indanone was formed in 62% enantiomeric excess (ee) (R:S, 81:19), while the 2-hydroxy-1-indanone was racemic. The same cells also formed 2-hydroxy-1-indanone from 2-indanone. Purified NDO components oxidized 1-indanone and 2-indanone to the same products produced by strain 9816/11. P. putida F39/D cells, expressing TDO, oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 76% ee (R:S, 12:88) but did not oxidize 1-indanone efficiently. Purified TDO components also oxidized 2-indanone to (S)-2-hydroxy-1-indanone of 90% ee (R:S, 5:95) and failed to oxidize 1-indanone. Oxidation of 1- and 2-indanone in the presence of [18O]oxygen indicated that the hydroxyindanones were formed by the incorporation of a single atom of molecular oxygen (monooxygenation) rather than by the dioxygenation of enol tautomers of the ketone substrates. As alternatives to chemical synthesis, these biotransformations represent direct routes to 3-hydroxy-1-indanone and 2-hydroxy-1-indanone as the major products from 1-indanone and 2-indanone, respectively. PMID:7944365

  3. Substrate Oxidation by Indoleamine 2,3-Dioxygenase

    PubMed Central

    Booth, Elizabeth S.; Basran, Jaswir; Lee, Michael; Handa, Sandeep; Raven, Emma L.

    2015-01-01

    The kynurenine pathway is the major route of l-tryptophan (l-Trp) catabolism in biology, leading ultimately to the formation of NAD+. The initial and rate-limiting step of the kynurenine pathway involves oxidation of l-Trp to N-formylkynurenine. This is an O2-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237–244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of l-Trp, 1-methyl-l-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues. PMID:26511316

  4. Molecular Dynamics Simulation of Nitrobenzene Dioxygenase Using AMBER Force Field

    PubMed Central

    2015-01-01

    Molecular dynamics simulation of the oxygenase component of nitrobenzene dioxygenase (NBDO) system, a member of the naphthalene family of Rieske nonheme iron dioxygenases, has been carried out using the AMBER force field combined with a new set of parameters for the description of the mononuclear nonheme iron center and iron–sulfur Rieske cluster. Simulation results provide information on the structure and dynamics of nitrobenzene dioxygenase in an aqueous environment and shed light on specific interactions that occur in its catalytic center. The results suggest that the architecture of the active site is stabilized by key hydrogen bonds, and Asn258 positions the substrate for oxidation. Analysis of protein–water interactions reveal the presence of a network of solvent molecules at the entrance to the active site, which could be of potential catalytic importance. PMID:24955078

  5. Assay and characterization of the NO dioxygenase activity of flavohemoglobins.

    PubMed

    Gardner, Paul R

    2008-01-01

    A variety of hemoglobins, including several microbial flavohemoglobins, enzymatically dioxygenate the free radical nitric oxide (*NO) to form nitrate. Many of these *NO dioxygenases have been shown to control *NO toxicity and signaling. Furthermore, *NO dioxygenation appears to be an ancient and intrinsic function for members of the hemoglobin superfamily found in Archaea, eukaryotes, and bacteria. Yet for many hemoglobins, a function remains to be elucidated. Methods for the assay and characterization of the *NO dioxygenase (EC 1.14.12.17) activity and function of flavohemoglobins are described. The methods may also be applied to the discovery and design of inhibitors for use as antibiotics or as modulators of *NO signaling.

  6. Mercaptosuccinate Dioxygenase, a Cysteine Dioxygenase Homologue, from Variovorax paradoxus Strain B4 Is the Key Enzyme of Mercaptosuccinate Degradation

    PubMed Central

    Brandt, Ulrike; Schürmann, Marc; Steinbüchel, Alexander

    2014-01-01

    The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent Km of 0.4 mm, and a specific activity (Vmax) of 20.0 μmol min−1 mg−1 corresponding to a kcat of 7.7 s−1. Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase. PMID:25228698

  7. Mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, from Variovorax paradoxus strain B4 is the key enzyme of mercaptosuccinate degradation.

    PubMed

    Brandt, Ulrike; Schürmann, Marc; Steinbüchel, Alexander

    2014-10-31

    The versatile thiol mercaptosuccinate has a wide range of applications, e.g. in quantum dot research or in bioimaging. Its metabolism is investigated in Variovorax paradoxus strain B4, which can utilize this compound as the sole source of carbon and sulfur. Proteomic studies of strain B4 resulted in the identification of a putative mercaptosuccinate dioxygenase, a cysteine dioxygenase homologue, possibly representing the key enzyme in the degradation of mercaptosuccinate. Therefore, the putative mercaptosuccinate dioxygenase was heterologously expressed, purified, and characterized in this study. The results clearly demonstrated that the enzyme utilizes mercaptosuccinate with concomitant consumption of oxygen. Thus, the enzyme is designated as mercaptosuccinate dioxygenase. Succinate and sulfite were verified as the final reaction products. The enzyme showed an apparent Km of 0.4 mM, and a specific activity (Vmax) of 20.0 μmol min(-1) mg(-1) corresponding to a kcat of 7.7 s(-1). Furthermore, the enzyme was highly specific for mercaptosuccinate, no activity was observed with cysteine, dithiothreitol, 2-mercaptoethanol, and 3-mercaptopropionate. These structurally related thiols did not have an inhibitory effect either. Fe(II) could clearly be identified as metal cofactor of the mercaptosuccinate dioxygenase with a content of 0.6 mol of Fe(II)/mol of enzyme. The recently proposed hypothesis for the degradation pathway of mercaptosuccinate based on proteome analyses could be strengthened in the present study. (i) Mercaptosuccinate is first converted to sulfinosuccinate by this mercaptosuccinate dioxygenase; (ii) sulfinosuccinate is spontaneously desulfinated to succinate and sulfite; and (iii) whereas succinate enters the central metabolism, sulfite is detoxified by the previously identified putative molybdopterin oxidoreductase.

  8. Substrate Stereo-specificity in Tryptophan dioxygenase and Indoleamine 2,3- dioxygenase

    PubMed Central

    Capece, L.; Arrar, M.; Roitberg, A. E.; Yeh, Syun-Ru; Marti, M. A.; Estrin, D. A.

    2010-01-01

    The first and rate-limiting step of the kynurenine pathway, in which tryptophan (Trp) is converted to N-formylkynurenine is catalyzed by two heme-containing proteins, Indoleamine 2,3-dioxygenase (IDO) and Tryptophan 2,3-dioxygenase (TDO). In mammals, TDO is found exclusively in liver tissue, IDO is found ubiquitously in all tissues. IDO has become increasingly popular in pharmaceutical research as it was found to be involved in many physiological situations, including immune escape of cancer. More importantly, small-molecule inhibitors of IDO are currently utilized in cancer therapy. One of the main concerns for the design of human IDO (hIDO) inhibitors is that they should be selective enough to avoid inhibition of TDO. In this work we have used a combination of classical molecular dynamics (MD) and hybrid quantum-classical (QM/MM) methodologies to establish the structural basis that determine the differences in a) the interactions of TDO and IDO with small ligands (CO/O2) and b) the substrate stereo-specificity in hIDO and TDO. Our results indicate that the differences in small ligand bound structures of IDO and TDO arise from slight differences in the structure of the bound substrate complex. The results also show that substrate stereo-specificity of TDO is achieved by the perfect fit of L-Trp, but not D-Trp, which exhibits weaker interactions with the protein matrix-. For hIDO, the presence of multiple stable binding conformations for L/D-Trp reveal the presence of a large and dynamic active site. Taken together, our data allow determination of key interactions useful for the future design of more potent hIDO-selective inhibitors. PMID:20715188

  9. Structure and Reaction Mechanism in the Heme Dioxygenases

    PubMed Central

    2011-01-01

    As members of the family of heme-dependent enzymes, the heme dioxygenases are differentiated by virtue of their ability to catalyze the oxidation of l-tryptophan to N-formylkynurenine, the first and rate-limiting step in tryptophan catabolism. In the past several years, there have been a number of important developments that have meant that established proposals for the reaction mechanism in the heme dioxygenases have required reassessment. This focused review presents a summary of these recent advances, written from a structural and mechanistic perspective. It attempts to present answers to some of the long-standing questions, to highlight as yet unresolved issues, and to explore the similarities and differences of other well-known catalytic heme enzymes such as the cytochromes P450, NO synthase, and peroxidases. PMID:21361337

  10. A Hyperactive Cobalt-Substituted Extradiol-Cleaving Catechol Dioxygenase

    PubMed Central

    Fielding, Andrew J.; Farquhar, Erik R.

    2011-01-01

    Homoprotocatechuate (HPCA) 2,3-dioxygenase from Brevibacterium fuscum (Fe-HPCD) has an Fe(II) center in its active site that can be replaced with Mn(II) or Co(II). While Mn-HPCD exhibits steady state kinetic parameters comparable to those of Fe-HPCD, Co-HPCD behaves somewhat differently exhibiting a significantly higher KMO2 and kcat. The high activity of Co-HPCD is surprising, given that cobalt has the highest standard M(III/II) redox potential of the three metals. Comparison of the X-ray crystal structures of the resting and substrate-bound forms of Fe-, Mn-, and Co-HPCD shows that metal-substitution has no effect on the local ligand environment, the conformational integrity of the active site, or the overall protein structure, suggesting that the protein structure does not differentially tune the potential of the metal center. Analysis of the steady state kinetics of Co-HPCD suggests that the Co(II) center alters the relative rate constants for the interconversion of intermediates in the catalytic cycle but still allows the dioxygenase reaction to proceed efficiently. When compared with the kinetic data for Fe- and Mn-HPCD, these results show that dioxygenase catalysis can proceed at high rates over a wide range of metal redox potentials. This is consistent with the proposed mechanism in which the metal mediates electron transfer between the catechol substrate and O2 to form the postulated [M(II)(semiquinone)superoxo] reactive species. These kinetic differences and the spectroscopic properties of Co-HPCD provide new tools with which to explore the unique O2 activation mechanism associated with the extradiol dioxygenase family. PMID:21153851

  11. Substrate Binding Mechanism of a Type I Extradiol Dioxygenase*

    PubMed Central

    Cho, Hyo Je; Kim, Kyungsun; Sohn, Seo Yean; Cho, Ha Yeon; Kim, Kyung Jin; Kim, Myung Hee; Kim, Dockyu; Kim, Eungbin; Kang, Beom Sik

    2010-01-01

    A meta-cleavage pathway for the aerobic degradation of aromatic hydrocarbons is catalyzed by extradiol dioxygenases via a two-step mechanism: catechol substrate binding and dioxygen incorporation. The binding of substrate triggers the release of water, thereby opening a coordination site for molecular oxygen. The crystal structures of AkbC, a type I extradiol dioxygenase, and the enzyme substrate (3-methylcatechol) complex revealed the substrate binding process of extradiol dioxygenase. AkbC is composed of an N-domain and an active C-domain, which contains iron coordinated by a 2-His-1-carboxylate facial triad motif. The C-domain includes a β-hairpin structure and a C-terminal tail. In substrate-bound AkbC, 3-methylcatechol interacts with the iron via a single hydroxyl group, which represents an intermediate stage in the substrate binding process. Structure-based mutagenesis revealed that the C-terminal tail and β-hairpin form part of the substrate binding pocket that is responsible for substrate specificity by blocking substrate entry. Once a substrate enters the active site, these structural elements also play a role in the correct positioning of the substrate. Based on the results presented here, a putative substrate binding mechanism is proposed. PMID:20810655

  12. Probes of the Catalytic Site of Cysteine Dioxygenase

    SciTech Connect

    Chai,S.; Bruyere, J.; Maroney, M.

    2006-01-01

    The first major step of cysteine catabolism, the oxidation of cysteine to cysteine sulfinic acid, is catalyzed by cysteine dioxygenase (CDO). In the present work, we utilize recombinant rat liver CDO and cysteine derivatives to elucidate structural parameters involved in substrate recognition and x-ray absorption spectroscopy to probe the interaction of the active site iron center with cysteine. Kinetic studies using cysteine structural analogs show that most are inhibitors and that a terminal functional group bearing a negative charge (e.g. a carboxylate) is required for binding. The substrate-binding site has no stringent restrictions with respect to the size of the amino acid. Lack of the amino or carboxyl groups at the a-carbon does not prevent the molecules from interacting with the active site. In fact, cysteamine is shown to be a potent activator of the enzyme without being a substrate. CDO was also rendered inactive upon complexation with the metal-binding inhibitors azide and cyanide. Unlike many non-heme iron dioxygenases that employ a-keto acids as cofactors, CDO was shown to be the only dioxygenase known to be inhibited by {alpha}-ketoglutarate.

  13. Biochemical Characterization of Inducible 'Reductase' Component of Benzoate Dioxygenase and Phthalate Isomer Dioxygenases from Pseudomonas aeruginosa strain PP4.

    PubMed

    Karandikar, Rohini; Badri, Abinaya; Phale, Prashant S

    2015-09-01

    The first step involved in the degradation of phthalate isomers (phthalate, isophthalate and terephthalate) is the double hydroxylation by respective aromatic-ring hydroxylating dioxygenases. These are two component enzymes consisting of 'oxygenase' and 'reductase' components. Soil isolate Pseudomonas aeruginosa strain PP4 degrades phthalate isomers via protocatechuate and benzoate via catechol 'ortho' ring cleavage pathway. Metabolic studies suggest that strain PP4 has carbon source-specific inducible phthalate isomer dioxygenase and benzoate dioxygenase. Thus, it was of interest to study the properties of reductase components of these enzymes. Reductase activity from phthalate isomer-grown cells was 3-5-folds higher than benzoate grown cells. In-gel activity staining profile showed a reductase activity band of R f 0.56 for phthalate isomer-grown cells as compared to R f 0.73 from benzoate-grown cells. Partially purified reductase components from phthalate isomer grown cells showed K m in the range of 30-40 μM and V max = 34-48 μmol min(-1) mg(-1). However, reductase from benzoate grown cells showed K m = 49 μM and V max = 10 μmol min(-1) mg(-1). Strikingly similar molecular and kinetic properties of reductase component from phthalate isomer-grown cells suggest that probably the same reductase component is employed in three phthalate isomer dioxygenases. However, reductase component is different, with respect to kinetic properties and zymogram analysis, from benzoate-grown cells when compared to that from phthalate isomer grown cells of PP4.

  14. Purification and characterization of protocatechuate 2,3-dioxygenase from Bacillus macerans: a new extradiol catecholic dioxygenase.

    PubMed Central

    Wolgel, S A; Dege, J E; Perkins-Olson, P E; Jaurez-Garcia, C H; Crawford, R L; Münck, E; Lipscomb, J D

    1993-01-01

    Protocatechuate 2,3-dioxygenase (2,3-PCD) from Bacillus macerans JJ1b has been purified to homogeneity for the first time. The enzyme catalyzes proximal extradiol ring cleavage of protocatechuate (PCA) with the attendant incorporation of both atoms of oxygen from O2. The holoenzyme has a mass of 143 +/- 7 kDa as determined by ultracentrifugation and other techniques. It is composed of four apparently identical subunits with M(r)s of 35,500, each containing one iron atom. Mössbauer spectroscopy of 57Fe-enriched enzyme showed that the irons are indistinguishable and are high spin (S = 2) Fe2+ in both the uncomplexed and substrate-bound enzyme. However, the quadrupole splitting, delta EQ, and isomer shift, delta, of the Mössbauer spectrum changed from delta EQ = 2.57 mm/s and delta = 1.29 mm/s to delta EQ = 2.73 mm/s and delta = 1.19 mm/s upon PCA binding to the enzyme, showing that the iron environment is altered when substrate is present. The enzyme was also found to bind variable and substoichiometric amounts of Mn2+, but this metal could be removed without loss of activity or stability. The inherently electron paramagnetic resonance (EPR)-silent Fe2+ of the enzyme reversibly bound nitric oxide to produce an EPR-active species (g = 4.11, 3.95; S = 3/2). The specific activity of the enzyme was found to be correlated with the amount of the S = 3/2 species formed, showing that activity is dependent on Fe2+. Anaerobic addition of substrates to the enzyme-nitric oxide complex significantly altered the EPR spectrum, suggesting that substrates bind to or near the iron. The enzyme was inactivated by reagents that oxidize the Fe2+, such as H2O2 and K3FE(CN)6; full activity was restored after reduction of the iron by ascorbate. Steady-state kinetic data were found to be consistent with an ordered bi-uni mechanism in which the organic substrate must add to 2,3-PCD before O2. The enzyme has the broadest substrate range of any of the well-studied catecholic dioxygenases. All

  15. Naphthalene dioxygenase: purification and properties of a terminal oxygenase component.

    PubMed Central

    Ensley, B D; Gibson, D T

    1983-01-01

    Naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816 is a multicomponent enzyme system that oxidized naphthalene to cis-(1R, 2S)-dihydroxy-1,2-dihydronaphthalene. The terminal oxygenase component B was purified to homogeneity by a three-step procedure that utilized ion-exchange and hydrophobic interaction chromatography. The purified enzyme oxidized naphthalene only in the presence of NADH, oxygen, and partially purified preparations of components A and C. An estimated Mr of 158,000 was obtained by gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of two subunits with molecular weights of ca. 55,000 and 20,000, indicative of an alpha 2 beta 2 quaternary structure. Absorption spectra of the oxidized enzyme showed maxima at 566 (shoulder), 462, and 344 nm, which were replaced by absorption maxima at 520 and 380 nm when the enzyme was reduced anaerobically by stoichiometric quantities of NADH in the presence of the other two components of the naphthalene dioxygenase system. Component B bound naphthalene. Enzyme-bound naphthalene was oxidized to product upon the addition of components A and C, NADH, and O2. These results, together with the detection of the presence of 6.0 g-atoms of iron and 4.0 g-atoms of acid-labile sulfur per mol of the purified enzyme, suggest that component B of the naphthalene dioxygenase system is an iron-sulfur protein which functions in the terminal step of naphthalene oxidation. PMID:6874638

  16. Comparative analysis of two DOPA dioxygenases from Phytolacca Americana.

    PubMed

    Takahashi, Kana; Yoshida, Kazuko; Sakuta, Masaaki

    2015-05-01

    The comparative analysis of two Phytolacca americana DOPA dioxygenases (PaDOD1 and PaDOD2) that may be involved in betalain biosynthesis was carried out. The recombinant protein of PaDOD catalyzed the conversion of DOPA to betalamic acid, whereas DOD activity was not detected in PaDOD2 in vitro. The role of DOD genes is discussed in the evolutionary context using phylogenetic analysis, suggesting that DOD might have been duplicated early in evolution and that accumulation of base substitutions could have led to the different characteristics of DODs within the betalain-producing Caryophyllales.

  17. Oxidative Transformation of Aminodinitrotoluene Isomers by Multicomponent Dioxygenases

    PubMed Central

    Johnson, Glenn R.; Smets, Barth F.; Spain, Jim C.

    2001-01-01

    The electron-withdrawing nitro substituents of 2,4,6-trinitrotoluene (TNT) make the aromatic ring highly resistant to oxidative transformation. The typical biological transformation of TNT involves reduction of one or more of the nitro groups of the ring to produce the corresponding amine. Reduction of a single nitro substituent of TNT to an amino substituent increases the electron density of the aromatic nucleus considerably. The comparatively electron-dense nuclei of the aminodinitrotoluene (ADNT) isomers would be expected to be more susceptible to oxygenase attack than TNT. The hypothesis was tested by evaluating three nitroarene dioxygenases for the ability to hydroxylate the ADNT isomers. The predominant reaction was dioxygenation of the ring to yield nitrite and the corresponding aminomethylnitrocatechol. A secondary reaction was benzylic monooxygenation to form aminodinitrobenzyl alcohol. The substrate preferences and catalytic specificities of the three enzymes differed considerably. The discovery that the ADNT isomers are substrates for the nitroarene dioxygenases reveals the potential for extensive bacterial transformation of TNT under aerobic conditions. PMID:11722893

  18. Distribution, Diversity, and Activities of Sulfur Dioxygenases in Heterotrophic Bacteria

    PubMed Central

    Liu, Honglei; Xin, Yufeng

    2014-01-01

    Sulfur oxidation by chemolithotrophic bacteria is well known; however, sulfur oxidation by heterotrophic bacteria is often ignored. Sulfur dioxygenases (SDOs) (EC 1.13.11.18) were originally found in the cell extracts of some chemolithotrophic bacteria as glutathione (GSH)-dependent sulfur dioxygenases. GSH spontaneously reacts with elemental sulfur to generate glutathione persulfide (GSSH), and SDOs oxidize GSSH to sulfite and GSH. However, SDOs have not been characterized for bacteria, including chemolithotrophs. The gene coding for human SDO (human ETHE1 [hETHE1]) in mitochondria was discovered because its mutations lead to a hereditary human disease, ethylmalonic encephalopathy. Using sequence analysis and activity assays, we discovered three subgroups of bacterial SDOs in the proteobacteria and cyanobacteria. Ten selected SDO genes were cloned and expressed in Escherichia coli, and the recombinant proteins were purified. The SDOs used Fe2+ for catalysis and displayed considerable variations in specific activities. The wide distribution of SDO genes reveals the likely source of the hETHE1 gene and highlights the potential of sulfur oxidation by heterotrophic bacteria. PMID:24389926

  19. A novel non-heme iron-containing dioxygenase. Chloridazon-catechol dioxygenase from Phenylobacterium immobilis DSM 1986.

    PubMed

    Müller, R; Schmitt, S; Lingens, F

    1982-07-01

    Previously we purified an enzyme from Phenylobacterium immobilis DSM 1986, which cleaves the catechol derivative of the herbicide Chloridazon [5-amino-4-chloro-2-phenyl-3 (2H)-pyridazinone] in the meta position. The enzyme, which could be crystallized, proved in Ouchterlony double-diffusion tests to consist of a single protein species. No cross-reaction was observed with other meta-cleaving enzymes. Its light absorption spectrum showed a maximum at 279 nm (epsilon = 310 mM -1 cm -1), shoulders at 289 nm and 275 nm and a very weak band at around 430 nm (epsilon = 1.14 mM -1 cm -1). The amino acid analysis showed a slight excess of acidic amino acids, in agreement with the pl of 4.5. Surprisingly the enzyme per se is completely inactive, although it contains one non-dialysable iron atom per submit. It has to be activated by preincubation with ferrous ions or ascorbate. The enzyme activated this way is autoxidizable and returns to its non-activated state in the presence of oxygen. During the reaction with the substrate, this inactivation seems to be enhanced about 100 times. Since this kind of activation and inactivation is not observed in other meta-cleaving enzymes, this enzyme seems to represent a new type of a non-heme iron dioxygenase. We tentatively propose the name Chloridazon-catechol dioxygenase for this enzyme.

  20. Molecules in focus: indoleamine 2,3-dioxygenase.

    PubMed

    King, Nicholas J C; Thomas, Shane R

    2007-01-01

    Indoleamine 2,3-dioxygenase (IDO) is a heme enzyme that initiates the oxidative degradation of the least abundant, essential amino acid, l-tryptophan, along the kynurenine pathway. The local cellular depletion of l-tryptophan that results may enable the host to inhibit the growth of various infectious pathogens in vivo. However, over the past decade, it has become increasingly apparent that IDO also represents an important immune control enzyme. Thus, cells expressing IDO, seemingly paradoxically, are capable of suppressing local T cell responses to promote immune tolerance under various physiological and pathophysiological conditions of medical importance, including infectious diseases, foetal rejection, organ transplantation, neuropathology, inflammatory and auto-immune disorders and cancer. In this review, we briefly outline the biochemical properties of IDO, its known and hypothetical functions and the medical implications for inhibition or induction of IDO and/or its downstream catabolites in health and disease.

  1. Ligand Migration in Human Indoleamine-2,3 Dioxygenase

    PubMed Central

    Nienhaus, Karin; Nickel, Elena; Lu, Changyuan; Yeh, Syun-Ru; Nienhaus, G. Ulrich

    2015-01-01

    Summary Human indoleamine 2,3-dioxygenase (hIDO), a monomeric heme enzyme, catalyzes the oxidative degradation of L-tryptophan (L-Trp) and other indoleamine derivatives. Its activity follows typical Michaelis–Menten behavior only for L-Trp concentrations up to 50 µM; a further increase in the concentration of L-Trp causes a decrease in the activity. This substrate inhibition of hIDO is a result of the binding of a second L-Trp molecule in an inhibitory substrate binding site of the enzyme. The molecular details of the reaction and the inhibition are not yet known. In the following, we summarize the present knowledge about this heme enzyme. PMID:21445845

  2. Molecular basis for the substrate stereoselectivity in Tryptophan Dioxygenase

    PubMed Central

    Capece, Luciana; Lewis-Ballester, Ariel; Marti, Marcelo A.; Estrin, Dario A.; Yeh, Syun-Ru

    2011-01-01

    Tryptophan dioxygenase (TDO) and Indoleamine 2,3 dioxygenase (IDO) are the only two heme-proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine (NFK). While human IDO (hIDO) is able to oxidize both L and D-Trp, human TDO (hTDO) displays a major specificity towards L-Trp. In this work we aim to interrogate the molecular basis for the substrate stereoselectivity of hTDO. Our previous molecular dynamics simulation studies of Xanthomonas campestris TDO (xcTDO) showed that an H-bond between T254 (T342 in hTDO) and the ammonium group of the substrate is present in the L-Trp-bound enzyme, but not in the D-Trp bound enzyme. The fact that this is the only notable structural alteration induced by the change in the stereo structure of the substrate prompted us to produce and characterize the T342A mutant of hTDO to evaluate the structural role of T342 in controlling the substrate stereoselectivity of the enzyme. The experimental results indicate that the mutation only slightly perturbs the global structural properties of the enzyme, but it totally abolishes the substrate stereoselectivity. Molecular Dynamics simulations of xcTDO show that T254 controls the substrate stereoselectivity of the enzyme by (i) modulating the H-bonding interaction between the NH3+ group and epoxide oxygen of the ferryl/indole 2,3-epoxide intermediate of the enzyme, and (ii) regulating the dynamics of two active site loops, loop250–260 and loop117–130, critical for substrate-binding. PMID:22082147

  3. INHIBITION OF INDOLEAMINE 2,3-DIOXYGENASE DOES NOT IMPEDE ORAL TOLERANCE

    EPA Science Inventory

    Rationale: Indoleamine 2,3-dioxygenase (IDO), a tryptophan catabolizing enzyme, regulates immune tolerance through inhibition of T-cell proliferation. Pharmacologic inhibition of IDO, which causes fetal rejection and increased tumor resistance in mice, may prove useful in cancer...

  4. Investigating the Role of Indoleamine 2,3-dioxygenase (IDO) in Breast Cancer Metastasis

    DTIC Science & Technology

    2011-09-01

    2,3-dioxygenase (IDO) in Breast Cancer Metastasis PRINCIPAL INVESTIGATOR: Courtney Smith, Ph.D...5a. CONTRACT NUMBER Investigating the Role of Indoleamine 2,3-dioxygenase (IDO) in Breast Cancer Metastasis 5b. GRANT NUMBER W81XWH-09-1-0667...the malignant 4T1 breast carcinoma cell line exhibit metastatic spread to organs similar to that seen in human breast cancer with pulmonary metastases

  5. Investigating the Role of Indoleamine 2,3- dioxygenase (IDO) in Breast Cancer Metastasis

    DTIC Science & Technology

    2012-09-01

    in Breast Cancer Metastasis” PRINCIPAL INVESTIGATOR: Courtney Smith, Ph.D. CONTRACTING ORGANIZATION: Lankenau Institute for Medical...4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER W Investigating the Role of Indoleamine 2,3-Dioxygenase (IDO) in Breast Cancer Metastasis 5b. GRANT...2,3-dioxygenase) has since been implicated in tumor escape from the host immune system. Primary tumor growth of the metastatic 4T1 breast cancer

  6. Properties of the iron--sulphur proteins of the benzene dioxygenase system from Pseudomonas putida.

    PubMed Central

    Crutcher, S E; Geary, P J

    1979-01-01

    A purification procedure was developed to stabilize the iron-sulphur proteins of the benzene dioxygenase system from Pseudomonas putida. The intermediate electron-carrying protein has a mol. wt. of 12300 and possesses one (2Fe--2S) cluster, whereas the terminal dioxygenase has a mol.wt. of 215300 and possesses two (2Fe--2S) clusters. The order and stoicheiometry of electron transfer and of the whole system are described. Images Fig. 2. PMID:435241

  7. Crystal Structure of PnpCD, a Two-subunit Hydroquinone 1,2-Dioxygenase, Reveals a Novel Structural Class of Fe2+-dependent Dioxygenases*

    PubMed Central

    Liu, Shiheng; Su, Tiantian; Zhang, Cong; Zhang, Wen-Mao; Zhu, Deyu; Su, Jing; Wei, Tiandi; Wang, Kang; Huang, Yan; Guo, Liming; Xu, Sujuan; Zhou, Ning-Yi; Gu, Lichuan

    2015-01-01

    Aerobic microorganisms have evolved a variety of pathways to degrade aromatic and heterocyclic compounds. However, only several classes of oxygenolytic fission reaction have been identified for the critical ring cleavage dioxygenases. Among them, the most well studied dioxygenases proceed via catecholic intermediates, followed by noncatecholic hydroxy-substituted aromatic carboxylic acids. Therefore, the recently reported hydroquinone 1,2-dioxygenases add to the diversity of ring cleavage reactions. Two-subunit hydroquinone 1,2-dioxygenase PnpCD, the key enzyme in the hydroquinone pathway of para-nitrophenol degradation, catalyzes the ring cleavage of hydroquinone to γ-hydroxymuconic semialdehyde. Here, we report three PnpCD structures, named apo-PnpCD, PnpCD-Fe3+, and PnpCD-Cd2+-HBN (substrate analog hydroxyenzonitrile), respectively. Structural analysis showed that both the PnpC and the C-terminal domains of PnpD comprise a conserved cupin fold, whereas PnpC cannot form a competent metal binding pocket as can PnpD cupin. Four residues of PnpD (His-256, Asn-258, Glu-262, and His-303) were observed to coordinate the iron ion. The Asn-258 coordination is particularly interesting because this coordinating residue has never been observed in the homologous cupin structures of PnpCD. Asn-258 is proposed to play a pivotal role in binding the iron prior to the enzymatic reaction, but it might lose coordination to the iron when the reaction begins. PnpD also consists of an intriguing N-terminal domain that might have functions other than nucleic acid binding in its structural homologs. In summary, PnpCD has no apparent evolutionary relationship with other iron-dependent dioxygenases and therefore defines a new structural class. The study of PnpCD might add to the understanding of the ring cleavage of dioxygenases. PMID:26304122

  8. Conversion of 3-Chlorocatechol by Various Catechol 2,3-Dioxygenases and Sequence Analysis of the Chlorocatechol Dioxygenase Region of Pseudomonas putida GJ31

    PubMed Central

    Mars, Astrid E.; Kingma, Jaap; Kaschabek, Stefan R.; Reineke, Walter; Janssen, Dick B.

    1999-01-01

    Pseudomonas putida GJ31 contains an unusual catechol 2,3-dioxygenase that converts 3-chlorocatechol and 3-methylcatechol, which enables the organism to use both chloroaromatics and methylaromatics for growth. A 3.1-kb region of genomic DNA of strain GJ31 containing the gene for this chlorocatechol 2,3-dioxygenase (cbzE) was cloned and sequenced. The cbzE gene appeared to be plasmid localized and was found in a region that also harbors genes encoding a transposase, a ferredoxin that was homologous to XylT, an open reading frame with similarity to a protein of a meta-cleavage pathway with unknown function, and a 2-hydroxymuconic semialdehyde dehydrogenase. CbzE was most similar to catechol 2,3-dioxygenases of the 2.C subfamily of type 1 extradiol dioxygenases (L. D. Eltis and J. T. Bolin, J. Bacteriol. 178:5930–5937, 1996). The substrate range and turnover capacity with 3-chlorocatechol were determined for CbzE and four related catechol 2,3-dioxygenases. The results showed that CbzE was the only enzyme that could productively convert 3-chlorocatechol. Besides, CbzE was less susceptible to inactivation by methylated catechols. Hybrid enzymes that were made of CzbE and the catechol 2,3-dioxygenase of P. putida UCC2 (TdnC) showed that the resistance of CbzE to suicide inactivation and its substrate specificity were mainly determined by the C-terminal region of the protein. PMID:9973359

  9. Evidence for a ferryl intermediate in a heme-based dioxygenase

    PubMed Central

    Lewis-Ballester, Ariel; Batabyal, Dipanwita; Egawa, Tsuyoshi; Lu, Changyuan; Lin, Yu; Marti, Marcelo A.; Capece, Luciana; Estrin, Dario A.; Yeh, Syun-Ru

    2009-01-01

    In contrast to the wide spectrum of cytochrome P450 monooxygenases, there are only 2 heme-based dioxygenases in humans: tryptophan dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). hTDO and hIDO catalyze the same oxidative ring cleavage reaction of L-tryptophan to N-formyl kynurenine, the initial and rate-limiting step of the kynurenine pathway. Despite immense interest, the mechanism by which the 2 enzymes execute the dioxygenase reaction remains elusive. Here, we report experimental evidence for a key ferryl intermediate of hIDO that supports a mechanism in which the 2 atoms of dioxygen are inserted into the substrate via a consecutive 2-step reaction. This finding introduces a paradigm shift in our understanding of the heme-based dioxygenase chemistry, which was previously believed to proceed via simultaneous incorporation of both atoms of dioxygen into the substrate. The ferryl intermediate is not observable during the hTDO reaction, highlighting the structural differences between the 2 dioxygenases, as well as the importance of stereoelectronic factors in modulating the reactions. PMID:19805032

  10. Engineering Non-Heme Mono- and Dioxygenases for Biocatalysis

    PubMed Central

    Dror, Adi; Fishman, Ayelet

    2012-01-01

    Oxygenases are ubiquitous enzymes that catalyze the introduction of one or two oxygen atoms to unreactive chemical compounds. They require reduction equivalents from NADH or NADPH and comprise metal ions, metal ion complexes, or coenzymes in their active site. Thus, for industrial purposes, oxygenases are most commonly employed using whole cell catalysis, to alleviate the need for co-factor regeneration. Biotechnological applications include bioremediation, chiral synthesis, biosensors, fine chemicals, biofuels, pharmaceuticals, food ingredients and polymers. Controlling activity and selectivity of oxygenases is therefore of great importance and of growing interest to the scientific community. This review focuses on protein engineering of non-heme monooxygenases and dioxygenases for generating improved or novel functionalities. Rational mutagenesis based on x-ray structures and sequence alignment, as well as random methods such as directed evolution, have been utilized. It is concluded that knowledge-based protein engineering accompanied with targeted libraries, is most efficient for the design and tuning of biocatalysts towards novel substrates and enhanced catalytic activity while minimizing the screening efforts. PMID:24688652

  11. Mechanism and Substrate Recognition of 2-Hydroxyethylphosphonate Dioxygenase

    PubMed Central

    2011-01-01

    HEPD belongs to the superfamily of 2-His-1-carboxylate non-heme iron-dependent dioxygenases. It converts 2-hydroxyethylphosphonate (2-HEP) to hydroxymethylphosphonate (HMP) and formate. Previously postulated mechanisms for the reaction catalyzed by HEPD cannot explain its conversion of 1-HEP to acetylphosphate. Alternative mechanisms that involve either phosphite or methylphosphonate as intermediates, which potentially explain all experimental studies including isotope labeling experiments and use of substrate analogues, were investigated. The results of these studies reveal that these alternative mechanisms are not correct. Site-directed mutagenesis studies of Lys16, Arg90, and Tyr98 support roles of these residues in binding of 2-HEP. Mutation of Lys16 to Ala resulted in an inactive enzyme, whereas mutation of Arg90 to Ala or Tyr98 to Phe greatly decreased kcat/Km,2-HEP. Furthermore, the latter mutants could not be saturated in O2. These results suggest that proper binding of 2-HEP is important for O2 activation and that the enzyme uses a compulsory binding order with 2-HEP binding before O2. The Y98F mutant produces methylphosphonate as a minor side product providing indirect support for the proposal that the last step during catalysis involves a ferric hydroxide reacting with a methylphosphonate radical. PMID:21711001

  12. Mechanism and Substrate Recognition of 2-Hydroxyethylphosphonate Dioxygenase

    SciTech Connect

    Peck, Spencer C.; Cooke, Heather A.; Cicchillo, Robert M.; Malova, Petra; Hammerschmidt, Friedrich; Nair, Satish K.; van der Donk, Wilfred A.

    2011-09-20

    HEPD belongs to the superfamily of 2-His-1-carboxylate non-heme iron-dependent dioxygenases. It converts 2-hydroxyethylphosphonate (2-HEP) to hydroxymethylphosphonate (HMP) and formate. Previously postulated mechanisms for the reaction catalyzed by HEPD cannot explain its conversion of 1-HEP to acetylphosphate. Alternative mechanisms that involve either phosphite or methylphosphonate as intermediates, which potentially explain all experimental studies including isotope labeling experiments and use of substrate analogues, were investigated. The results of these studies reveal that these alternative mechanisms are not correct. Site-directed mutagenesis studies of Lys16, Arg90, and Tyr98 support roles of these residues in binding of 2-HEP. Mutation of Lys16 to Ala resulted in an inactive enzyme, whereas mutation of Arg90 to Ala or Tyr98 to Phe greatly decreased k{sub cat}/K{sub m,2-HEP}. Furthermore, the latter mutants could not be saturated in O{sub 2}. These results suggest that proper binding of 2-HEP is important for O{sub 2} activation and that the enzyme uses a compulsory binding order with 2-HEP binding before O{sub 2}. The Y98F mutant produces methylphosphonate as a minor side product providing indirect support for the proposal that the last step during catalysis involves a ferric hydroxide reacting with a methylphosphonate radical.

  13. Differential Expression of Cysteine Dioxygenase 1 in Complex Karyotype Liposarcomas

    PubMed Central

    Shaker, Mohammed; Pascarelli, Kara M; Plantinga, Matthew J; Love, Miles A; Lazar, Alexander J; Ingram, Davis R; von Mehren, Margaret; Lev, Dina; Kipling, David; Broccoli, Dominique

    2014-01-01

    Altered cysteine dioxygenase 1 (CDO1) gene expression has been observed in several cancers but has not yet been investigated in liposarcomas. The aim of this study was to evaluate CDO1 expression in a cohort of liposarcomas and to determine its association with clinicopathological features. Existing microarray data indicated variable CDO1 expression in liposarcoma subtypes. CDO1 mRNA from a larger cohort of liposarcomas was quantified by real time-PCR, and CDO1 protein expression was determined by immunohistochemistry (IHC) in more than 300 tumor specimens. Well-differentiated liposarcomas (WDLSs) had significantly higher CDO1 gene expression and protein levels than dedifferentiated liposarcomas (DDLSs) (P < 0.001). Location of the tumor was not predictive of the expression level of CDO1 mRNA in any histological subtype of liposarcoma. Recurrent tumors did not show any difference in CDO1 expression when compared to primary tumors. CDO1 expression was upregulated as human mesenchymal stem cells (hMSCs) undergo differentiation into mature adipocytes. Our results suggest that CDO1 is a marker of liposarcoma progression and adipogenic differentiation. PMID:24741338

  14. Antitumour agents as inhibitors of tryptophan 2,3-dioxygenase

    SciTech Connect

    Pantouris, Georgios; Mowat, Christopher G.

    2014-01-03

    Highlights: •∼2800 National Cancer Institute USA compounds have been screened as potential inhibitors of TDO and/or IDO. •Seven compounds with anti-tumour properties have been identified as potent inhibitors. •NSC 36398 (taxifolin, dihydroquercetin) is selective for TDO with a K{sub i} of 16 M. •This may help further our understanding of the role of TDO in cancer. -- Abstract: The involvement of tryptophan 2,3-dioxygenase (TDO) in cancer biology has recently been described, with the enzyme playing an immunomodulatory role, suppressing antitumour immune responses and promoting tumour cell survival and proliferation. This finding reinforces the need for specific inhibitors of TDO that may potentially be developed for therapeutic use. In this work we have screened ∼2800 compounds from the library of the National Cancer Institute USA and identified seven potent inhibitors of TDO with inhibition constants in the nanomolar or low micromolar range. All seven have antitumour properties, killing various cancer cell lines. For comparison, the inhibition potencies of these compounds were tested against IDO and their inhibition constants are reported. Interestingly, this work reveals that NSC 36398 (dihydroquercetin, taxifolin), with an in vitro inhibition constant of ∼16 μM, is the first TDO-selective inhibitor reported.

  15. 3-mercaptopropionate dioxygenase, a cysteine dioxygenase homologue, catalyzes the initial step of 3-mercaptopropionate catabolism in the 3,3-thiodipropionic acid-degrading bacterium variovorax paradoxus.

    PubMed

    Bruland, Nadine; Wübbeler, Jan Hendrik; Steinbüchel, Alexander

    2009-01-02

    The thioether 3,3-thiodipropionic acid can be used as precursor substrate for biotechnological synthesis of 3-mercaptopropionic acid-containing polythioesters. Therefore, the hitherto unknown catabolism of this compound was elucidated to engineer novel and improved polythioester biosynthesis pathways in the future. Bacteria capable of using 3,3-thiodipropionic acid as the sole source of carbon and energy for growth were enriched from the environment. From eleven isolates, TBEA3, TBEA6, and SFWT were morphologically and physiologically characterized. Their 16 S rDNAs and other features affiliated these isolates to the beta-subgroup of the proteobacteria. Tn5::mob mutagenesis of isolate Variovorax paradoxus TBEA6 yielded ten mutants fully or partially impaired in growth on 3,3-thiodipropionic acid. Genotypic characterization of two 3,3-thiodipropionic acid-negative mutants demonstrated the involvement of a bacterial cysteine dioxygenase (EC 1.13.11.22) homologue in the further catabolism of the 3,3-thiodipropionic acid cleavage product 3-mercaptopropionic acid. Detection of 3-sulfinopropionate in the supernatant of one of these mutants during cultivation on 3,3-thiodipropionic acid as well as in vivo and in vitro enzyme assays using purified protein demonstrated oxygenation of 3-mercaptopropionic acid to 3-sulfinopropionate by this enzyme; cysteine and cysteamine were not used as substrate. Beside cysteine dioxygenase and cysteamine dioxygenase, this 3-mercaptopropionic acid dioxygenase is the third example for a thiol dioxygenase and the first report about the microbial catabolism of 3-mercaptopropionic acid. Insertion of Tn5::mob in a gene putatively coding for a family III acyl-CoA-transferase resulted in the accumulation of 3-sulfinopropionate during cultivation on 3,3-thiodipropionic acid, indicating that this compound is further metabolized to 3-sulfinopropionyl-CoA and subsequently to propionyl-CoA.

  16. Expression, purification and kinetic characterization of recombinant benzoate dioxygenase from Rhodococcus ruber UKMP-5M

    PubMed Central

    Tavakoli, Arezoo; Hamzah, Ainon; Rabu, Amir

    2016-01-01

    In this study, benzoate dioxygenase from Rhodococcus ruber UKMP-5M was catalyzed by oxidating the benzene ring to catechol and other derivatives. The benzoate dioxygenase (benA gene) from Rhodococcus ruber UKMP-5M was then expressed, purified, characterized, The benA gene was amplified (642 bp), and the product was cloned into a pGEM-T vector. The recombinant plasmid pGEMT-benA was digested by double restriction enzymes BamHI and HindIII to construct plasmid pET28b-benA and was then ligated into Escherichia coli BL21 (DE3). The recombinant E. coli was induced with 0.5 mM isopropyl β-D-thiogalactoside (IPTG) at 22˚C to produce benzoate dioxygenase. The enzyme was then purified by ion exchange chromatography after 8 purification folds. The resulting product was 25 kDa, determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. Benzoate dioxygenase activity was found to be 6.54 U/mL and the optimal pH and temperature were 8.5 and 25°C, respectively. Maximum velocity (Vmax) and Michaelis constant (Km) were 7.36 U/mL and 5.58 µM, respectively. The end metabolite from the benzoate dioxygenase reaction was cyclohexane dione, which was determined by gas chromatography mass spectrometry (GC-MS). PMID:28097167

  17. Chloridazon-catechol dioxygenases, a distinct group of meta-cleaving enzymes.

    PubMed

    Schmitt, S; Müller, R; Wegst, W; Lingens, F

    1984-02-01

    We previously described a new meta-cleaving enzyme, termed chloridazon-catechol dioxygenase. The present paper describes the comparison of this enzyme with the meta-cleaving enzymes of eighteen strains of soil bacteria isolated with various aromatic compounds. Four of these strains were isolated with the herbicide chloridazon, six with the analgeticum aminopyrine and one with the analgeticum antipyrine as sole carbon source. These strains all belonged to a new type of bacteria, called Phenylobacteria. The seven other strains were isolated with aromatic compounds such as toluene, 3-phenylpropionate, benzoate, papaverine and 4-chlorobenzoate, and belonged to various species including Pseudomonas, Acinetobacter and Nocardia. In double diffusion experiments with antibodies, prepared against chloridazon-catechol dioxygenase, extracts from the eleven strains of Phenylobacteria gave a cross reaction, whereas the extracts of the seven other strains showed no reaction. The enzymes of the eleven positive strains showed the same characteristic kinetic behaviour as the previously described enzyme. In contrast to catechol 2, 3-dioxygenase they needed the addition of exogenous Fe2+ ions for activity. On ion-exchange chromatography they emerged at the same buffer concentration as chloridazon-catechol dioxygenase. In polyacrylamide electrophoresis they migrated identically. The linkage map derived from the activities of the various enzymes with 10 different substrates revealed an identity of more than 80% for these eleven enzymes. So the meta-cleaving enzymes of the Phenylobacteria seem to form a distinct group among the non-heme iron-containing dioxygenases.

  18. Substrate Oxidation by Indoleamine 2,3-Dioxygenase: EVIDENCE FOR A COMMON REACTION MECHANISM.

    PubMed

    Booth, Elizabeth S; Basran, Jaswir; Lee, Michael; Handa, Sandeep; Raven, Emma L

    2015-12-25

    The kynurenine pathway is the major route of L-tryptophan (L-Trp) catabolism in biology, leading ultimately to the formation of NAD(+). The initial and rate-limiting step of the kynurenine pathway involves oxidation of L-Trp to N-formylkynurenine. This is an O2-dependent process and catalyzed by indoleamine 2,3-dioxygenase and tryptophan 2,3-dioxygenase. More than 60 years after these dioxygenase enzymes were first isolated (Kotake, Y., and Masayama, I. (1936) Z. Physiol. Chem. 243, 237-244), the mechanism of the reaction is not established. We examined the mechanism of substrate oxidation for a series of substituted tryptophan analogues by indoleamine 2,3-dioxygenase. We observed formation of a transient intermediate, assigned as a Compound II (ferryl) species, during oxidation of L-Trp, 1-methyl-L-Trp, and a number of other substrate analogues. The data are consistent with a common reaction mechanism for indoleamine 2,3-dioxygenase-catalyzed oxidation of tryptophan and other tryptophan analogues.

  19. Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR

    SciTech Connect

    Mesarch, M.B.; Nakatsu, C.H.; Nies, L.

    2000-02-01

    Benzene, toluene, xylenes, phenol, naphthalene, and biphenyl are among a group of compounds that have at least one reported pathway for biodegradation involving catechol 2,3-dioxygenase enzymes. Thus, detection of the corresponding catechol 2,3-dioxygenase genes can serve as a basis for identifying and quantifying bacteria that have these catabolic abilities. Primes that can successfully amplify a 238-bp catechol 2,3-dioxygenase gene fragment from eight different bacteria are described. The identities of the amplicons were confirmed by hybridization with a 238-bp catechol 2,3-dioxygenase probe. The detection limit was 10{sup 2} to 10{sup 3} gene copies, which was lowered to 10{sup 0} to 10{sup 1} gene copies of hybridization. Using the dioxygenase-specific primers, an increase in catechol 2,3-dioxygenase genes was detected in petroleum-amended soils. The dioxygenase genes were enumerated by competitive quantitative PCR and a 163-bp competitor that was amplified using the same primers. Target and competitor sequences had identical amplification kinetics. Potential PCR inhibitors that could coextract with DNA, nonamplifying DNA, soil factors (humics), and soil pollutants (toluene) did not impact enumeration. Therefore, this technique can be used to accurately and reproducibly quantify catechol 2,3-dioxygenase genes in complex environments such as petroleum-contaminated soil. Direct, non-cultivation-based molecular techniques for detecting and enumerating microbial pollutant-biodegrading genes in environmental samples are powerful tools for monitoring bioremediation and developing field evidence in support of natural attenuation.

  20. Substrate specificities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenase enzyme systems.

    PubMed

    Parales, R E; Emig, M D; Lynch, N A; Gibson, D T

    1998-05-01

    Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (alpha and beta). To assess the contributions of the alpha and beta subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different beta subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.

  1. Metal-dependent function of a mammalian acireductone dioxygenase

    PubMed Central

    Deshpande, Aditi R.; Wagenpfeil, Karina; Pochapsky, Thomas C.; Petsko, Gregory A.; Ringe, Dagmar

    2017-01-01

    The two acireductone dioxygenase (ARD) isozymes from the methionine salvage pathway of Klebsiella oxytoca are the only known pair of naturally occurring metalloenzymes with distinct chemical and physical properties determined solely by the identity of the divalent transition metal ion (Fe2+ or Ni2+) in the active site. We now show that this dual chemistry can also occur in mammals. ARD from Mus musculus (MmARD) was studied to relate metal ion identity and three-dimensional structure to enzyme function. The iron-containing isozyme catalyzes the cleavage of 1,2-dihydroxy-3-keto-5-(thiomethyl)pent-1-ene (acireductone) by O2 to formate and the ketoacid precursor of methionine, the penultimate step in methionine salvage. The nickel bound form of ARD catalyzes an off-pathway reaction resulting in formate, carbon monoxide (CO) and 5-(thiomethyl) propionate. Recombinant MmARD was expressed and purified to obtain a homogeneous enzyme with a single transition metal ion bound. The Fe2+ bound protein, which shows about ten-fold higher activity than others, catalyzes on-pathway chemistry, whereas the Ni2+, Co2+ or Mn2+ forms exhibit off-pathway chemistry, as has been seen with ARD from Klebsiella. Thermal stability of the isozymes is strongly affected by metal ion identity, with Ni2+ bound MmARD being the most stable followed by Co2+ and Fe2+, and Mn2+-bound ARD being the least stable. Ni2+ and Co2+ bound MmARD were crystallized and the structures of the two proteins found to be similar. Enzyme-ligand complexes provide insight into substrate binding, metal coordination and catalytic mechanism. PMID:26858196

  2. Molecular Cloning, Nucleotide Sequence, and Expression of Genes Encoding a Polycyclic Aromatic Ring Dioxygenase from Mycobacterium sp. Strain PYR-1

    PubMed Central

    Khan, Ashraf A.; Wang, Rong-Fu; Cao, Wei-Wen; Doerge, Daniel R.; Wennerstrom, David; Cerniglia, Carl E.

    2001-01-01

    Mycobacterium sp. strain PYR-1 degrades high-molecular-weight polycyclic hydrocarbons (PAHs) primarily through the introduction of both atoms of molecular oxygen by a dioxygenase. To clone the dioxygenase genes involved in PAH degradation, two-dimensional (2D) gel electrophoresis of PAH-induced proteins from cultures of Mycobacterium sp. strain PYR-1 was used to detect proteins that increased after phenanthrene, dibenzothiophene, and pyrene exposure. Comparison of proteins from induced and uninduced cultures on 2D gels indicated that at least six major proteins were expressed (105, 81, 52, 50, 43, and 13 kDa). The N-terminal sequence of the 50-kDa protein was similar to those of other dioxygenases. A digoxigenin-labeled oligonucleotide probe designed from this protein sequence was used to screen dioxygenase-positive clones from a genomic library of Mycobacterium sp. strain PYR-1. Three clones, each containing a 5,288-bp DNA insert with three genes of the dioxygenase system, were obtained. The genes in the DNA insert, from the 5′ to the 3′ direction, were a dehydrogenase, the dioxygenase small (β)-subunit, and the dioxygenase large (α)-subunit genes, arranged in a sequence different from those of genes encoding other bacterial dioxygenase systems. Phylogenetic analysis showed that the large α subunit did not cluster with most of the known α-subunit sequences but rather with three newly described α subunits of dioxygenases from Rhodococcus spp. and Nocardioides spp. The genes from Mycobacterium sp. strain PYR-1 were subcloned and overexpressed in Escherichia coli with the pBAD/ThioFusion system. The functionality of the genes for PAH degradation was confirmed in a phagemid clone containing all three genes, as well as in plasmid subclones containing the two genes encoding the dioxygenase subunits. PMID:11472934

  3. Expression pattern and localization of beta,beta-carotene 15,15'-dioxygenase in different tissues.

    PubMed Central

    Wyss, A; Wirtz, G M; Woggon, W D; Brugger, R; Wyss, M; Friedlein, A; Riss, G; Bachmann, H; Hunziker, W

    2001-01-01

    Beta,beta-carotene 15,15'-dioxygenase cleaves beta,beta-carotene into two molecules of retinal, and is the key enzyme in the metabolism of beta,beta-carotene to vitamin A. The enzyme has been known for more than 40 years, yet all attempts to purify the protein to homogeneity have failed. Recently, the successful cloning and sequencing of an enzyme with beta,beta-carotene 15,15'-dioxygenase activity from chicken, as well as from Drosophila, has been reported. Here, we describe in detail our attempt to enrich the chicken beta,beta-carotene 15,15'-dioxygenase to such an extent as to allow determination of partial amino acid sequences, which were then used to design degenerate oligonucleotides. Screening of a chicken duodenal expression library yielded a full-length clone containing a coding sequence of 1578 bp. Functional expression in Escherichia coli and in eukaryotic cell lines confirmed that we had cloned the first vertebrate dioxygenase that cleaves beta,beta-carotene at the central 15,15'-double bond. By performing a sequence homology search, the cDNA sequence of the mouse homologue was found as an expressed sequence tag (EST) in the gene bank. At the amino-acid level, the degree of homology between the chicken and mouse sequences is 81%. Thus beta,beta-carotene 15,15'-dioxygenase can be considered as being an enzyme that is evolutionarily rather well conserved. We established the expression pattern of beta,beta-carotene 15,15'-dioxygenase in chicken and mouse tissues with a combination of Northern blots and in situ hybridization. The mRNA for beta,beta-carotene 15,15'-dioxygenase was localized primarily in duodenal villi, as well as in liver and in tubular structures of lung and kidney. These new findings demonstrate that beta,beta-carotene 15,15'-dioxygenase is also expressed in epithelial structures, where it serves to provide the tissue-specific vitamin A supply. PMID:11237856

  4. Enzymology of the carotenoid cleavage dioxygenases: reaction mechanisms, inhibition and biochemical roles.

    PubMed

    Harrison, Peter J; Bugg, Timothy D H

    2014-02-15

    Carotenoid cleavage dioxygenases (CCDs) are a large family of non-heme iron (II) dependent enzymes. CCDs catalyse the selective oxidative cleavage of carotenoids to produce apocarotenoids. Apocarotenoid derived molecules form important signalling molecules in plants in the form of abscisic acid and strigolactone and in mammals in the form of retinal. Very little is known biochemically about the CCDs and only a handful of CCDs have been biochemically characterised. Mechanistically, debate surrounds whether CCDs utilise a mono or dioxygenase mechanism. Here, we review the biochemical roles of CCDs, discuss the mechanisms by which CCD cleavage is proposed to occur, and discuss recent reports of selective CCD enzyme inhibitors.

  5. The first step of the dioxygenation reaction carried out by tryptophan dioxygenase and indoleamine 2,3-dioxygenase as revealed by quantum mechanical/molecular mechanical studies

    PubMed Central

    Capece, Luciana; Lewis-Ballester, Ariel; Batabyal, Dipanwita; Di Russo, Natali; Estrin, Dario A.

    2015-01-01

    Tryptophan dioxygenase (TDO) and indole-amine 2,3-dioxygenase (IDO) are two heme-containing enzymes which catalyze the conversion of L-tryptophan to N-formylkynurenine (NFK). In mammals, TDO is mostly expressed in liver and is involved in controlling homeostatic serum tryptophan concentrations, whereas IDO is ubiquitous and is involved in modulating immune responses. Previous studies suggested that the first step of the dioxygenase reaction involves the deprotonation of the indoleamine group of the substrate by an evolutionarily conserved distal histidine residue in TDO and the heme-bound dioxygen in IDO. Here, we used classical molecular dynamics and hybrid quantum mechanical/molecular mechanical methods to evaluate the base-catalyzed mechanism. Our data suggest that the deprotonation of the indoleamine group of the substrate by either histidine in TDO or heme-bound dioxygen in IDO is not energetically favorable. Instead, the dioxygenase reaction can be initiated by a direct attack of heme-bound dioxygen on the C2=C3 bond of the indole ring, leading to a protein-stabilized 2,3-alkylperoxide transition state and a ferryl epoxide intermediate, which subsequently recombine to generate NFK. The novel sequential two-step oxygen addition mechanism is fully supported by our recent resonance Raman data that allowed identification of the ferryl intermediate (Lewis-Ballester et al. in Proc Natl Acad Sci USA 106:17371–17376, 2009). The results reveal the subtle differences between the TDO and IDO reactions and highlight the importance of protein matrix in modulating stereoelectronic factors for oxygen activation and the stabilization of both transition and intermediate states. PMID:20361220

  6. Steady-state substrate specificity and O₂-coupling efficiency of mouse cysteine dioxygenase.

    PubMed

    Li, Wei; Pierce, Brad S

    2015-01-01

    Cysteine dioxygenase (CDO) is a non-heme mononuclear iron enzyme that catalyzes the oxygen-dependent oxidation of L-cysteine (Cys) to produce L-cysteine sulfinic acid (CSA). Sequence alignment of mammalian CDO with recently discovered thiol dioxygenase enzymes suggests that the mononuclear iron site within all enzymes in this class share a common 3-His first coordination sphere. This implies a similar mechanistic paradigm among thiol dioxygenase enzymes. Although steady-state studies were first reported for mammalian CDO over 45 years ago, detailed analysis of the specificity for alternative thiol-bearing substrates and their oxidative coupling efficiencies have not been reported for this enzyme. Assuming a similar mechanistic theme among this class of enzymes, characterization of the CDO substrate specificity may provide valuable insight into substrate-active site intermolecular during thiol oxidation. In this work, the substrate-specificity for wild-type Mus musculus CDO was investigated using NMR spectroscopy and LC-MS for a variety of thiol-bearing substrates. Tandem mass spectrometry was used to confirm dioxygenase activity for each non-native substrate investigated. Steady-state Michaelis-Menten parameters for sulfinic acid product formation and O₂-consumption were compared to establish the coupling efficiency for each reaction. In light of these results, the minimal substrate requirements for CDO catalysis and O₂-activation are discussed.

  7. An iron-oxygen intermediate formed during the catalytic cycle of cysteine dioxygenase.

    PubMed

    Tchesnokov, E P; Faponle, A S; Davies, C G; Quesne, M G; Turner, R; Fellner, M; Souness, R J; Wilbanks, S M; de Visser, S P; Jameson, G N L

    2016-07-07

    Cysteine dioxygenase is a key enzyme in the breakdown of cysteine, but its mechanism remains controversial. A combination of spectroscopic and computational studies provides the first evidence of a short-lived intermediate in the catalytic cycle. The intermediate decays within 20 ms and has absorption maxima at 500 and 640 nm.

  8. Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase

    SciTech Connect

    Knoot, Cory J.; Purpero, Vincent M.; Lipscomb, John D.

    2014-12-29

    Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe3+ to activate O2 and catecholic substrates for reaction. The inability of Fe3+ to directly bind O2 presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated in this paper using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies. When the reaction was initiated in an enzyme crystal, it was found to halt at one of two intermediates depending on the pH of the surrounding solution. The X-ray crystal structure of the intermediate at pH 6.5 revealed the key alkylperoxo-Fe3+ species, and the anhydride-Fe3+ intermediate was found for a crystal reacted at pH 8.5. Intermediates of these types have not been structurally characterized for intradiol dioxygenases, and they validate four decades of spectroscopic, kinetic, and computational studies. In contrast to our similar in crystallo crystallographic studies of an Fe2+-containing extradiol dioxygenase, no evidence for a superoxo or peroxo intermediate preceding the alkylperoxo was found. This observation and the lack of spectroscopic evidence for an Fe2+ intermediate that could bind O2 are consistent with concerted formation of the alkylperoxo followed by Criegee rearrangement to yield the anhydride and ultimately ring-opened product. Finally, structural comparison of the alkylperoxo intermediates from the intra- and extradiol dioxygenases provides a rationale for site specificity of ring cleavage.

  9. Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase

    DOE PAGES

    Knoot, Cory J.; Purpero, Vincent M.; Lipscomb, John D.

    2014-12-29

    Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe3+ to activate O2 and catecholic substrates for reaction. The inability of Fe3+ to directly bind O2 presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated in this paper using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies. When the reaction was initiated in an enzyme crystal, it was found to halt at one of two intermediates depending on the pH of the surrounding solution. The X-ray crystalmore » structure of the intermediate at pH 6.5 revealed the key alkylperoxo-Fe3+ species, and the anhydride-Fe3+ intermediate was found for a crystal reacted at pH 8.5. Intermediates of these types have not been structurally characterized for intradiol dioxygenases, and they validate four decades of spectroscopic, kinetic, and computational studies. In contrast to our similar in crystallo crystallographic studies of an Fe2+-containing extradiol dioxygenase, no evidence for a superoxo or peroxo intermediate preceding the alkylperoxo was found. This observation and the lack of spectroscopic evidence for an Fe2+ intermediate that could bind O2 are consistent with concerted formation of the alkylperoxo followed by Criegee rearrangement to yield the anhydride and ultimately ring-opened product. Finally, structural comparison of the alkylperoxo intermediates from the intra- and extradiol dioxygenases provides a rationale for site specificity of ring cleavage.« less

  10. Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase

    PubMed Central

    Knoot, Cory J.; Purpero, Vincent M.; Lipscomb, John D.

    2015-01-01

    Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe3+ to activate O2 and catecholic substrates for reaction. The inability of Fe3+ to directly bind O2 presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated here using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies. When the reaction was initiated in an enzyme crystal, it was found to halt at one of two intermediates depending on the pH of the surrounding solution. The X-ray crystal structure of the intermediate at pH 6.5 revealed the key alkylperoxo-Fe3+ species, and the anhydride-Fe3+ intermediate was found for a crystal reacted at pH 8.5. Intermediates of these types have not been structurally characterized for intradiol dioxygenases, and they validate four decades of spectroscopic, kinetic, and computational studies. In contrast to our similar in crystallo crystallographic studies of an Fe2+-containing extradiol dioxygenase, no evidence for a superoxo or peroxo intermediate preceding the alkylperoxo was found. This observation and the lack of spectroscopic evidence for an Fe2+ intermediate that could bind O2 are consistent with concerted formation of the alkylperoxo followed by Criegee rearrangement to yield the anhydride and ultimately ring-opened product. Structural comparison of the alkylperoxo intermediates from the intra- and extradiol dioxygenases provides a rationale for site specificity of ring cleavage. PMID:25548185

  11. Crystal structures of alkylperoxo and anhydride intermediates in an intradiol ring-cleaving dioxygenase.

    PubMed

    Knoot, Cory J; Purpero, Vincent M; Lipscomb, John D

    2015-01-13

    Intradiol aromatic ring-cleaving dioxygenases use an active site, nonheme Fe(3+) to activate O2 and catecholic substrates for reaction. The inability of Fe(3+) to directly bind O2 presents a mechanistic conundrum. The reaction mechanism of protocatechuate 3,4-dioxygenase is investigated here using the alternative substrate 4-fluorocatechol. This substrate is found to slow the reaction at several steps throughout the mechanistic cycle, allowing the intermediates to be detected in solution studies. When the reaction was initiated in an enzyme crystal, it was found to halt at one of two intermediates depending on the pH of the surrounding solution. The X-ray crystal structure of the intermediate at pH 6.5 revealed the key alkylperoxo-Fe(3+) species, and the anhydride-Fe(3+) intermediate was found for a crystal reacted at pH 8.5. Intermediates of these types have not been structurally characterized for intradiol dioxygenases, and they validate four decades of spectroscopic, kinetic, and computational studies. In contrast to our similar in crystallo crystallographic studies of an Fe(2+)-containing extradiol dioxygenase, no evidence for a superoxo or peroxo intermediate preceding the alkylperoxo was found. This observation and the lack of spectroscopic evidence for an Fe(2+) intermediate that could bind O2 are consistent with concerted formation of the alkylperoxo followed by Criegee rearrangement to yield the anhydride and ultimately ring-opened product. Structural comparison of the alkylperoxo intermediates from the intra- and extradiol dioxygenases provides a rationale for site specificity of ring cleavage.

  12. Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems.

    PubMed

    Parales, R E; Huang, R; Yu, C-L; Parales, J V; Lee, F K N; Lessner, D J; Ivkovic-Jensen, M M; Liu, W; Friemann, R; Ramaswamy, S; Gibson, D T

    2005-07-01

    The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.

  13. Purification, Characterization, and Crystallization of the Components of the Nitrobenzene and 2-Nitrotoluene Dioxygenase Enzyme Systems

    PubMed Central

    Parales, R. E.; Huang, R.; Yu, C.-L.; Parales, J. V.; Lee, F. K. N.; Lessner, D. J.; Ivkovic-Jensen, M. M.; Liu, W.; Friemann, R.; Ramaswamy, S.; Gibson, D. T.

    2005-01-01

    The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an α3β3 hexamer. The apparent Km of 2-nitrotoluene dioxygenase for 2NT was 20 μM, and that for naphthalene was 121 μM. The specificity constants were 7.0 μM−1 min−1 for 2NT and 1.2 μM−1 min−1 for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained. PMID:16000792

  14. Purification and properties of ferredoxinNAP, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

    PubMed Central

    Haigler, B E; Gibson, D T

    1990-01-01

    One of the three components of the naphthalene dioxygenase occurring in induced cells of Pseudomonas sp. strain NCIB 9816 has been purified to homogeneity. The protein contained 2 g-atoms each of iron and acid-labile sulfur and had an apparent molecular weight of 13,600. The evidence indicates that it is a ferredoxin-type protein that functions as an intermediate electron transfer protein in naphthalene dioxygenase activity. PMID:2294093

  15. Assessment of toluene/biphenyl dioxygenase gene diversity in benzene-polluted soils: links between benzene biodegradation and genes similar to those encoding isopropylbenzene dioxygenases.

    PubMed

    Witzig, Robert; Junca, Howard; Hecht, Hans-Jürgen; Pieper, Dietmar H

    2006-05-01

    The PCR-single-strand conformation polymorphism (SSCP) technique was used to assess the diversity and distribution of Rieske nonheme iron oxygenases of the toluene/biphenyl subfamily in soil DNA and bacterial isolates recovered from sites contaminated with benzene, toluene, ethylbenzene, and xylenes (BTEX). The central cores of genes encoding the catalytic alpha subunits were targeted, since they are responsible for the substrate specificities of these enzymes. SSCP functional genotype fingerprinting revealed a substantial diversity of oxygenase genes in three differently BTEX-contaminated soil samples, and sequence analysis indicated that in both the soil DNA and the bacterial isolates, genes for oxygenases related to the isopropylbenzene (cumene) dioxygenase branch of the toluene/biphenyl oxygenase subfamily were predominant among the detectable genotypes. The peptide sequences of the two most abundant alpha subunit sequence types differed by only five amino acids (residues 258, 286, 288, 289, and 321 according to numbering in cumene dioxygenase alpha subunit CumA1 of Pseudomonas fluorescens IP01). However, a strong correlation between sequence type and substrate utilization pattern was observed in isolates harboring these genes. Two of these residues were located at positions contributing, according to the resolved crystal structure of cumene dioxygenase from Pseudomonas fluorescens IP01, to the inner surface of the substrate-binding pocket. Isolates containing an alpha subunit with isoleucine and leucine at positions 288 and 321, respectively, were capable of degrading benzene and toluene, whereas isolates containing two methionine substitutions were found to be incapable of degrading toluene, indicating that the more bulky methionine residues significantly narrowed the available space within the substrate-binding pocket.

  16. X-ray structures of 4-chlorocatechol 1,2-dioxygenase adducts with substituted catechols: new perspectives in the molecular basis of intradiol ring cleaving dioxygenases specificity.

    PubMed

    Ferraroni, Marta; Kolomytseva, Marina; Scozzafava, Andrea; Golovleva, Ludmila; Briganti, Fabrizio

    2013-03-01

    The crystallographic structures of 4-chlorocatechol 1,2-dioxygenase (4-CCD) complexes with 3,5-dichlorocatechol, protocatechuate (3,4-dihydroxybenzoate), hydroxyquinol (benzen-1,2,4-triol) and pyrogallol (benzen-1,2,3-triol), which act as substrates or inhibitors of the enzyme, have been determined and analyzed. 4-CCD from the Gram-positive bacterium Rhodococcus opacus 1CP is a Fe(III) ion containing enzyme specialized in the aerobic biodegradation of chlorocatechols. The structures of the 4-CCD complexes show that the catechols bind the catalytic iron ion in a bidentate mode displacing Tyr169 and the benzoate ion (found in the native enzyme structure) from the metal coordination sphere, as found in other adducts of intradiol dioxygenases with substrates. The analysis of the present structures allowed to identify the residues selectively involved in recognition of the diverse substrates. Furthermore the structural comparison with the corresponding complexes of catechol 1,2-dioxygenase from the same Rhodococcus strain (Rho-1,2-CTD) highlights significant differences in the binding of the tested catechols to the active site of the enzyme, particularly in the orientation of the aromatic ring substituents. As an example the 3-substituted catechols are bound with the substituent oriented towards the external part of the 4-CCD active site cavity, whereas in the Rho-1,2-CTD complexes the 3-substituents were placed in the internal position. The present crystallographic study shed light on the mechanism that allows substrate recognition inside this class of high specific enzymes involved in the biodegradation of recalcitrant pollutants.

  17. Crystal structure of 3-hydroxyanthranilic acid 3,4-dioxygenase from Saccharomyces cerevisiae: a special subgroup of the type III extradiol dioxygenases.

    PubMed

    Li, Xiaowu; Guo, Min; Fan, Jun; Tang, Wenying; Wang, Deqiang; Ge, Honghua; Rong, Hui; Teng, Maikun; Niu, Liwen; Liu, Qun; Hao, Quan

    2006-04-01

    3-Hydroxyanthranilic acid 3,4-dioxygenase (3HAO) is a non-heme ferrous extradiol dioxygenase in the kynurenine pathway from tryptophan. It catalyzes the conversion of 3-hydroxyanthranilate (HAA) to quinolinic acid (QUIN), an endogenous neurotoxin, via the activation of N-methyl-D-aspartate (NMDA) receptors and the precursor of NAD(+) biosynthesis. The crystal structure of 3HAO from S. cerevisiae at 2.4 A resolution shows it to be a member of the functionally diverse cupin superfamily. The structure represents the first eukaryotic 3HAO to be resolved. The enzyme forms homodimers, with two nickel binding sites per molecule. One of the bound nickel atoms occupies the proposed ferrous-coordinated active site, which is located in a conserved double-strand beta-helix domain. Examination of the structure reveals the participation of a series of residues in catalysis different from other extradiol dioxygenases. Together with two iron-binding residues (His49 and Glu55), Asp120, Asn51, Glu111, and Arg114 form a hydrogen-bonding network; this hydrogen-bond network is key to the catalysis of 3HAO. Residues Arg101, Gln59, and the substrate-binding hydrophobic pocket are crucial for substrate specificity. Structure comparison with 3HAO from Ralstonia metallidurans reveals similarities at the active site and suggests the same catalytic mechanism in prokaryotic and eukaryotic 3HAO. Based on sequence comparison, we suggest that bicupin of human 3HAO is the first example of evolution from a monocupin dimer to bicupin monomer in the diverse cupin superfamilies. Based on the model of the substrate HAA at the active site of Y3HAO, we propose a mechanism of catalysis for 3HAO.

  18. Cloning, expression, and characterization of catechol 1,2-dioxygenase from a phenol-degrading Candida tropicalis JH8 strain.

    PubMed

    Long, Yan; Yang, Sheng; Xie, Zhixiong; Cheng, Li

    2016-10-02

    The sequence cato encoding catechol 1,2-dioxygenase from Candida tropicalis JH8 was cloned, sequenced, and expressed in Escherichia coli. The sequence cato contained an ORF of 858 bp encoding a polypeptide of 285 amino acid residues. The recombinant catechol 1,2-dioxygenase exists as a homodimer structure with a subunit molecular mass of 32 KD. Recombinant catechol 1,2-dioxygenase was unstable below pH 5.0 and stable from pH 7.0 to 9.0; its optimum pH was at 7.5. The optimum temperature for the enzyme was 30°C, and it possessed a thermophilic activity within a broad temperature range. Under the optimal conditions with catechol as substrate, the Km and Vmax of recombinant catechol 1,2-dioxygenase were 9.2 µM and 0.987 µM/min, respectively. This is the first article presenting cloning and expressing in E. coli of catechol 1,2-dioxygenase from C. tropicalis and characterization of the recombinant catechol 1,2-dioxygenase.

  19. Characterization of catechol 2,3-dioxygenase from Planococcus sp. strain S5 induced by high phenol concentration.

    PubMed

    Hupert-Kocurek, Katarzyna; Guzik, Urszula; Wojcieszyńska, Danuta

    2012-01-01

    This study aimed at characterization of a new catechol 2,3-dioxygenase isolated from a Gram-positive bacterium able to utilize phenol as the sole carbon and energy source. Planococcus sp. strain S5 grown on 1 or 2 mM phenol showed activity of both a catechol 1,2- and catechol 2,3-dioxygenase while at a higher concentrations of phenol only catechol 2,3-dioxygenase activity was observed. The enzyme was optimally active at 60°C and pH 8.0. Kinetic studies showed that the K(m) and V(max) of the enzyme were 42.70 µM and 329.96 mU, respectively. The catechol 2,3-dioxygenase showed the following relative meta-cleavage activities for various catechols tested: catechol (100%), 3-methylcatechol (13.67%), 4-methylcatechol (106.33%) and 4-chlorocatechol (203.80%). The high reactivity of this enzyme towards 4-chlorocatechol is different from that observed for other catechol 2,3-dioxygenases. Nucleotide sequencing and homology search revealed that the gene encoding the S5 catechol 2,3-dioxygenase shared the greatest homology with the known genes encoding isoenzymes from Gram-negative Pseudomonas strains.

  20. A two-electron shell game: Intermediates of the extradiol-cleaving catechol dioxygenases

    PubMed Central

    Fielding, Andrew J.

    2014-01-01

    Extradiol catechol ring-cleaving dioxygenases function by binding both the organic substrate and O2 at a divalent metal center in the active site. They have proven to be a particularly versatile group of enzymes with which to study the O2 activation process. Here, recent studies of homoprotocatechuate 2,3-dioxygenase (HPCD) are summarized with the objective of showing how Nature can utilize the enzyme structure and the properties of the metal and the substrate to select among many possible chemical paths to achieve both specificity and efficiency. Possible intermediates in the mechanism have been trapped by swapping active site metals, introducing active site amino acid substituted variants, and using substrates with different electron donating capacities. While each of these intermediates could form part of a viable reaction pathway, kinetic measurements significantly limit the likely candidates. Structural, kinetic, spectroscopic and computational analysis of the various intermediates shed light on how catalytic efficiency can be achieved. PMID:24615282

  1. Trichloroethylene degradation by Escherichia coli containing the cloned Pseudomonas putida F1 toluene dioxygenase genes

    SciTech Connect

    Zylstra, G.J.; Gibson, D.T. ); Wackett, L.P. )

    1989-12-01

    Toluene dioxygenase from Pseudomonas putida F1 has been implicated as an enzyme capable of degrading trichloroethylene. This has now been confirmed with Escherichia coli JM109(pDTG601) that contains the structural genes (todC1C2BA) of toluene dioxygenase under the control of the tac promoter. The extent of trichloroethylene degradation by the recombinant organism depended on the cell concentration and the concentration of trichloroethylene. A linear rate of trichloroethylene degradation was observed with the E. coli recombinant strain. In contrast, P. putida F39/D, a mutant strain of P. putida F1 that does not contain cis-toluene dihydrodiol dehydrogenase, showed a much faster initial rate of trichloroethylene degradation which decreased over time.

  2. Oxidation of nitrotoluenes by toluene dioxygenase: evidence for a monooxygenase reaction.

    PubMed

    Robertson, J B; Spain, J C; Haddock, J D; Gibson, D T

    1992-08-01

    Pseudomonas putida F1 and Pseudomonas sp. strain JS150 initiate toluene degradation by incorporating molecular oxygen into the aromatic nucleus to form cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene. When toluene-grown cells were incubated with 2- and 3-nitrotoluene, the major products identified were 2- and 3-nitrobenzyl alcohol, respectively. The same cells oxidized 4-nitrotoluene to 2-methyl-5-nitrophenol and 3-methyl-6-nitrocatechol. Escherichia coli JM109(pDTG601), which contains the toluene dioxygenase genes from P. putida F1 under the control of the tac promoter, oxidized the isomeric nitrotoluenes to the same metabolites as those formed by P. putida F1 and Pseudomonas sp. strain JS150. These results extend the range of substrates known to be oxidized by this versatile enzyme and demonstrate for the first time that toluene dioxygenase can oxidize an aromatic methyl substituent.

  3. Origin and evolution of peptide-modifying dioxygenases and identification of the wybutosine hydroxylase/hydroperoxidase

    PubMed Central

    Iyer, Lakshminarayan M.; Abhiman, Saraswathi; de Souza, Robson F.; Aravind, L.

    2010-01-01

    Unlike classical 2-oxoglutarate and iron-dependent dioxygenases, which include several nucleic acid modifiers, the structurally similar jumonji-related dioxygenase superfamily was only known to catalyze peptide modifications. Using comparative genomics methods, we predict that a family of jumonji-related enzymes catalyzes wybutosine hydroxylation/peroxidation at position 37 of eukaryotic tRNAPhe. Identification of this enzyme raised questions regarding the emergence of protein- and nucleic acid-modifying activities among jumonji-related domains. We addressed these with a natural classification of DSBH domains and reconstructed the precursor of the dioxygenases as a sugar-binding domain. This precursor gave rise to sugar epimerases and metal-binding sugar isomerases. The sugar isomerase active site was exapted for catalysis of oxygenation, with a radiation of these enzymes in bacteria, probably due to impetus from the primary oxygenation event in Earth’s history. 2-Oxoglutarate-dependent versions appear to have further expanded with rise of the tricarboxylic acid cycle. We identify previously under-appreciated aspects of their active site and multiple independent innovations of 2-oxoacid-binding basic residues among these superfamilies. We show that double-stranded β-helix dioxygenases diversified extensively in biosynthesis and modification of halogenated siderophores, antibiotics, peptide secondary metabolites and glycine-rich collagen-like proteins in bacteria. Jumonji-related domains diversified into three distinct lineages in bacterial secondary metabolism systems and these were precursors of the three major clades of eukaryotic enzymes. The specificity of wybutosine hydroxylase/peroxidase probably relates to the structural similarity of the modified moiety to the ancestral amino acid substrate of this superfamily. PMID:20423905

  4. Unravelling the Molecular Origin of the Regiospecificity in Extradiol Catechol Dioxygenases.

    PubMed

    Christian, Gemma J; Neese, Frank; Ye, Shengfa

    2016-04-18

    Many factors have been suggested to control the selectivity for extradiol or intradiol cleavage in catechol dioxygenases. The varied selectivity of model complexes and the ability to force an extradiol enzyme to do intradiol cleavage indicate that the problem may be complex. In this paper we focus on the regiospecificity of the proximal extradiol dioxygenase, homoprotocatechuate 2,3-dioxygenase (HPCD), for which considerable advances have been made in our understanding of the mechanism from an experimental and computational standpoint. Two key steps in the reaction mechanism were investigated: (1) attack of the substrate by the superoxide moiety and (2) attack of the substrate by the oxyl radical generated by O-O bond cleavage. The selectivity at both steps was investigated through a systematic study of the role of the substrate and the first and second coordination spheres. For the isolated native substrate, intradiol cleavage is calculated to be both kinetically and thermodynamically favored, therefore nature must use the enzyme environment to reverse this preference. Two second sphere residues were found to play key roles in controlling the regiospecificity of the reaction: Tyr257 and His200. Tyr257 controls the selectivity by modulating the electronic structure of the substrate, while His200 controls selectivity through steric effects and by preventing alternative pathways to intradiol cleavage.

  5. [Isolation, charcaterization of an anthracene degrading bacterium Martelella sp. AD-3 and cloning of dioxygenase gene].

    PubMed

    Cui, Chang-Zheng; Feng, Tian-Cai; Yu, Ya-Qi; Dong, Fei; Yang, Xin-Mei; Feng, Yao-Yu; Liu, Yong-Di; Lin, Han-Ping

    2012-11-01

    Anthracene, among the 16 US EPA polycyclic aromatic hydrocarbons (PAHs), is a typical low molecular weight environmental contaminant, which gains concern on its biodegradation under hypersaline condition. In this study, an anthracene-degrading bacterial strain was isolated from highly saline petroleum-contaminated soil. Based on its physiological, biochemical characteristics and 16S rDNA sequence analysis, the bacteria was preliminary identified and named as Martelella sp. AD-3. The strain was able to utilize anthracene as sole carbon source for growth and the degradation occurred under broad salinities (0.1% to 10%) and varying pHs (6.0 to 10.0). The optimized degradation conditions were initial concentration 25 mg x L(-1), culture temperature 30 degrees C, pH 9.0 and salinity 3%. And 94.6% of anthracene was degraded by strain AD-3 under the optimal conditions within 6 days. Degenerate primers design was performed with a reported dioxygenase alpha subunit homologous gene. A length of 307 bp fragment of the partial dioxygenase gene sequences (GenBank accession: JF823991.1) was amplified by nested PCR. The clones amino acid sequence from strain AD-3 showed 95% identity to that of the partial naphthalene dioxygenase large-subunit from Marinobacter sp. NCE312 (AF295033). The results lay a foundation for the further study of molecular mechanism involved in the PAHs biodegradation by strain AD-3.

  6. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs.

    PubMed

    Driggers, Camden M; Hartman, Steven J; Karplus, P Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ∼15-30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue ("Arg-type" enzymes) and some having a Gln substituted for this Arg ("Gln-type" enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis "Arg-type" enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha "Gln-type" CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among "Gln-type" CDO enzymes, we conclude that the "Gln-type" CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

  7. The Mechanism of Formation of N-Formylkynurenine by Heme Dioxygenases

    PubMed Central

    2011-01-01

    Heme dioxygenases catalyze the oxidation of l-tryptophan to N-formylkynurenine (NFK), the first and rate-limiting step in tryptophan catabolism. Although recent progress has been made on early stages in the mechanism, there is currently no experimental data on the mechanism of product (NFK) formation. In this work, we have used mass spectrometry to examine product formation in a number of dioxygenases. In addition to NFK formation (m/z = 237), the data identify a species (m/z = 221) that is consistent with insertion of a single atom of oxygen into the substrate during O2-driven turnover. The fragmentation pattern for this m/z = 221 species is consistent with a cyclic amino acetal structure; independent chemical synthesis of the 3a-hydroxypyrroloindole-2-carboxylic acid compound is in agreement with this assignment. Labeling experiments with 18O2 confirm the origin of the oxygen atom as arising from O2-dependent turnover. These data suggest that the dioxygenases use a ring-opening mechanism during NFK formation, rather than Criegee or dioxetane mechanisms as previously proposed. PMID:21892828

  8. Characterization and evolution of vertebrate indoleamine 2, 3-dioxygenases IDOs from monotremes and marsupials.

    PubMed

    Yuasa, Hajime J; Ball, Helen J; Ho, Yuen Fern; Austin, Christopher J D; Whittington, Camilla M; Belov, Katherine; Maghzal, Ghassan J; Jermiin, Lars S; Hunt, Nicholas H

    2009-06-01

    Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in tryptophan catabolism via the kynurenine pathway. TDO is widely distributed in both eukaryotes and bacteria. In contrast, IDO has been found only in mammals and yeast. In 2007, a third enzyme, indoleamine 2,3-dioxygenase-2 (IDO2), was discovered. IDO2 is found not only in mammals but also in lower vertebrates. Interestingly, the Km value of IDO2 for L-Trp was 500-1000 fold higher than that of IDO1. In this study, we isolated both IDO1 and IDO2 cDNA from a monotreme, the platypus (Ornithorhynchus anatinus), and a marsupial, the gray short-tailed opossum (Monodelphis domestica). We characterized the recombinant proteins and those of other known IDO1/IDO2 in intact cells and a cell-free system. It was found that methylene blue may not be suitable reductant for IDO2, hence resulting in an underestimation of recombinant IDO2 activity. In intact cells, the Km value of IDO2 for L-Trp was estimated to be much higher than that of IDO1 and this high Km value appears to have been conserved during the evolution of IDO2. The protein encoded by the ancestor gene of IDO1 and IDO2 is likely to have had properties more similar to present day IDO2 than to IDO1.

  9. Characterization and evolution of vertebrate indoleamine 2, 3-dioxygenases IDOs from monotremes and marsupials.

    PubMed

    Yuasa, Hajime J; Ball, Helen J; Ho, Yuen Fern; Austin, Christopher J D; Whittington, Camilla M; Belov, Katherine; Maghzal, Ghassan J; Jermiin, Lars S; Hunt, Nicholas H

    2009-06-01

    Indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in tryptophan catabolism via the kynurenine pathway. TDO is widely distributed in both eukaryotes and bacteria. In contrast, IDO has been found only in mammals and yeast. In 2007, a third enzyme, indoleamine 2,3-dioxygenase-2 (IDO2), was discovered. IDO2 is found not only in mammals but also in lower vertebrates. Interestingly, the K(m) value of IDO2 for L-Trp was 500-1000 fold higher than that of IDO1. In this study, we isolated both IDO1 and IDO2 cDNA from a monotreme, the platypus (Ornithorhynchus anatinus), and a marsupial, the gray short-tailed opossum (Monodelphis domestica). We characterized the recombinant proteins and those of other known IDO1/IDO2 in intact cells and a cell-free system. It was found that methylene blue may not be suitable reductant for IDO2, hence resulting in an underestimation of recombinant IDO2 activity. In intact cells, the K(m) value of IDO2 for L-Trp was estimated to be much higher than that of IDO1 and this high K(m) value appears to have been conserved during the evolution of IDO2. The protein encoded by the ancestor gene of IDO1 and IDO2 is likely to have had properties more similar to present day IDO2 than to IDO1.

  10. Application of nitroarene dioxygenases in the design of novel strains that degrade chloronitrobenzenes

    PubMed Central

    Ju, Kou‐San; Parales, Rebecca E.

    2009-01-01

    Summary Widespread application of chloronitrobenzenes as feedstocks for the production of industrial chemicals and pharmaceuticals has resulted in extensive environmental contamination with these toxic compounds, where they pose significant risks to the health of humans and wildlife. While biotreatment in general is an attractive solution for remediation, its effectiveness is limited with chloronitrobenzenes due to the small number of strains that can effectively mineralize these compounds and their ability to degrade only select isomers. To address this need, we created engineered strains with a novel degradation pathway that reduces the total number of steps required to convert chloronitrobenzenes into compounds of central metabolism. We examined the ability of 2‐nitrotoluene 2,3‐dioxygenase from Acidovorax sp. strain JS42, nitrobenzene 1,2‐dioxygenase (NBDO) from Comamonas sp. strain JS765, as well as active‐site mutants of NBDO to generate chlorocatechols from chloronitrobenzenes, and identified the most efficient enzymes. Introduction of the wild‐type NBDO and the F293Q variant into Ralstonia sp. strain JS705, a strain carrying the modified ortho pathway for chlorocatechol metabolism, resulted in bacterial strains that were able to sustainably grow on all three chloronitrobenzene isomers without addition of co‐substrates or co‐inducers. These first‐generation engineered strains demonstrate the utility of nitroarene dioxygenases in expanding the metabolic capabilities of bacteria and provide new options for improved biotreatment of chloronitrobenzene‐contaminated sites. PMID:21261918

  11. Characterizations of Two Bacterial Persulfide Dioxygenases of the Metallo-β-lactamase Superfamily*

    PubMed Central

    Sattler, Steven A.; Wang, Xia; Lewis, Kevin M.; DeHan, Preston J.; Park, Chung-Min; Xin, Yufeng; Liu, Honglei; Xian, Ming; Xun, Luying; Kang, ChulHee

    2015-01-01

    Persulfide dioxygenases (PDOs), also known as sulfur dioxygenases (SDOs), oxidize glutathione persulfide (GSSH) to sulfite and GSH. PDOs belong to the metallo-β-lactamase superfamily and play critical roles in animals, plants, and microorganisms, including sulfide detoxification. The structures of two PDOs from human and Arabidopsis thaliana have been reported; however, little is known about the substrate binding and catalytic mechanism. The crystal structures of two bacterial PDOs from Pseudomonas putida and Myxococcus xanthus were determined at 1.5- and 2.5-Å resolution, respectively. The structures of both PDOs were homodimers, and their metal centers and β-lactamase folds were superimposable with those of related enzymes, especially the glyoxalases II. The PDOs share similar Fe(II) coordination and a secondary coordination sphere-based hydrogen bond network that is absent in glyoxalases II, in which the corresponding residues are involved instead in coordinating a second metal ion. The crystal structure of the complex between the Pseudomonas PDO and GSH also reveals the similarity of substrate binding between it and glyoxalases II. Further analysis implicates an identical mode of substrate binding by known PDOs. Thus, the data not only reveal the differences in metal binding and coordination between the dioxygenases and the hydrolytic enzymes in the metallo-β-lactamase superfamily, but also provide detailed information on substrate binding by PDOs. PMID:26082492

  12. Oxidation of aliphatic olefins by toluene dioxygenase: enzyme rates and product identification.

    PubMed Central

    Lange, C C; Wackett, L P

    1997-01-01

    Toluene dioxygenase from Pseudomonas putida F1 has been studied extensively with aromatic substrates. The present work examined the toluene dioxygenase-catalyzed oxidation of various halogenated ethenes, propenes, butenes and nonhalogenated cis-2-pentene, an isomeric mix of 2-hexenes, cis-2-heptene, and cis-2-octene as substrates for toluene dioxygenase. Enzyme specific activities were determined for the more water-soluble C2 to C5 compounds and ranged from <4 to 52 nmol per min per mg of protein. Trichloroethene was oxidized at a rate of 33 nmol per min per mg of protein. Products from enzyme reactions were identified by gas chromatography-mass spectrometry. Proton and carbon nuclear magnetic resonance spectroscopy of compounds from whole-cell incubation confirmed the identity of products. Substrates lacking a halogen substituent on sp2 carbon atoms were dioxygenated, while those with halogen and one or more unsubstituted allylic methyl groups were monooxygenated to yield allylic alcohols. 2,3-Dichloro-1-propene, containing both a halogenated double bond and a halogenated allylic methyl group, underwent monooxygenation with allylic rearrangement to yield an isomeric mixture of cis- and trans-2,3-dichloro-2-propene-1-ol. PMID:9190800

  13. Crystal structure of the terminal oxygenase component of cumene dioxygenase from Pseudomonas fluorescens IP01.

    PubMed

    Dong, Xuesong; Fushinobu, Shinya; Fukuda, Eriko; Terada, Tohru; Nakamura, Shugo; Shimizu, Kentaro; Nojiri, Hideaki; Omori, Toshio; Shoun, Hirofumi; Wakagi, Takayoshi

    2005-04-01

    The crystal structure of the terminal component of the cumene dioxygenase multicomponent enzyme system of Pseudomonas fluorescens IP01 (CumDO) was determined at a resolution of 2.2 A by means of molecular replacement by using the crystal structure of the terminal oxygenase component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4 (NphDO). The ligation of the two catalytic centers of CumDO (i.e., the nonheme iron and Rieske [2Fe-2S] centers) and the bridging between them in neighboring catalytic subunits by hydrogen bonds through a single amino acid residue, Asp231, are similar to those of NphDO. An unidentified external ligand, possibly dioxygen, was bound at the active site nonheme iron. The entrance to the active site of CumDO is different from the entrance to the active site of NphDO, as the two loops forming the lid exhibit great deviation. On the basis of the complex structure of NphDO, a biphenyl substrate was modeled in the substrate-binding pocket of CumDO. The residues surrounding the modeled biphenyl molecule include residues that have already been shown to be important for its substrate specificity by a number of engineering studies of biphenyl dioxygenases.

  14. Characterization of hbzE-encoded gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Yeo, Chew Chieng; Tan, Chew Ling; Gao, Xiaoli; Zhao, Bing; Poh, Chit Laa

    2007-09-01

    Pseudomonas alcaligenes NCIMB 9867 (strain P25X) is known to synthesize two isofunctional gentisate 1,2-dioxygenases (GDO; EC 1.13.11.4) as well as other enzymes involved in the degradation of xylenols and cresols via the gentisate pathway. The hbzE gene encoding what is possibly the strictly inducible gentisate 1,2-dioxygenase II (GDO-II) was cloned, overexpressed and purified as a hexahistidine fusion protein from Escherichia coli. Active recombinant GDO-II had an estimated molecular mass of 150kDa and is likely a tetrameric protein with a subunit mass of approximately 40kDa, similar to the previously characterized gentisate 1,2-dioxygenase I (GDO-I) encoded by xlnE. However, GDO-II was unable to utilize gentisate that is substituted at the carbon-4 position, unlike GDO-I which had broader substrate specificity. GDO-II also possessed different kinetic characteristics when compared to GDO-I. The hbzE-encoded GDO-II shared higher sequence identities (53%) with GDOs from Ralstonia sp. U2 and Polaromonas naphthalenivorans CJ2, compared with only 35% identity with the xlnE-encoded GDO-I. The hbzE gene was found to be part of a cluster of nine genes including the putative regulatory gene designated hbzR, which encodes an LysR-type regulator and is divergently transcribed from the other genes of the hbzHIJKLFED cluster.

  15. Isolation and characterization of two novel halotolerant Catechol 2, 3-dioxygenases from a halophilic bacterial consortium

    PubMed Central

    Guo, Guang; Fang, Tingting; Wang, Chongyang; Huang, Yong; Tian, Fang; Cui, Qijia; Wang, Hui

    2015-01-01

    Study of enzymes in halophiles will help to understand the mechanism of aromatic hydrocarbons degradation in saline environment. In this study, two novel catechol 2,3-dioxygenases (C23O1 and C23O2) were cloned and overexpressed from a halophilic bacterial consortium enriched from an oil-contaminated saline soil. Phylogenetic analysis indicated that the novel C23Os and their relatives formed a new branch in subfamily I.2.A of extradiol dioxygenases and the sequence differences were further analyzed by amino acid sequence alignment. Two enzymes with the halotolerant feature were active over a range of 0–30% salinity and they performed more stable at high salinity than in the absence of salt. Surface electrostatic potential and amino acids composition calculation suggested high acidic residues content, accounting for their tolerance to high salinity. Moreover, two enzymes were further characterized. The enzymes activity both increased in the presence of Fe3+, Fe2+, Cu2+ and Al3+ and showed no significant inhibition by other tested metal ions. The optimal temperatures for the C23Os were 40 °C and 60 °C and their best substrates were catechol and 4-methylcatechol respectively. As the firstly isolated and characterized catechol dioxygenases from halophiles, the two halotolerant C23Os presented novel characteristics suggesting their potential application in aromatic hydrocarbons biodegradation. PMID:26621792

  16. Isolation and characterization of two novel halotolerant Catechol 2, 3-dioxygenases from a halophilic bacterial consortium

    NASA Astrophysics Data System (ADS)

    Guo, Guang; Fang, Tingting; Wang, Chongyang; Huang, Yong; Tian, Fang; Cui, Qijia; Wang, Hui

    2015-12-01

    Study of enzymes in halophiles will help to understand the mechanism of aromatic hydrocarbons degradation in saline environment. In this study, two novel catechol 2,3-dioxygenases (C23O1 and C23O2) were cloned and overexpressed from a halophilic bacterial consortium enriched from an oil-contaminated saline soil. Phylogenetic analysis indicated that the novel C23Os and their relatives formed a new branch in subfamily I.2.A of extradiol dioxygenases and the sequence differences were further analyzed by amino acid sequence alignment. Two enzymes with the halotolerant feature were active over a range of 0-30% salinity and they performed more stable at high salinity than in the absence of salt. Surface electrostatic potential and amino acids composition calculation suggested high acidic residues content, accounting for their tolerance to high salinity. Moreover, two enzymes were further characterized. The enzymes activity both increased in the presence of Fe3+, Fe2+, Cu2+ and Al3+ and showed no significant inhibition by other tested metal ions. The optimal temperatures for the C23Os were 40 °C and 60 °C and their best substrates were catechol and 4-methylcatechol respectively. As the firstly isolated and characterized catechol dioxygenases from halophiles, the two halotolerant C23Os presented novel characteristics suggesting their potential application in aromatic hydrocarbons biodegradation.

  17. Enzyme Reactivation by Hydrogen Peroxide in Heme-based Tryptophan Dioxygenase*

    PubMed Central

    Fu, Rong; Gupta, Rupal; Geng, Jiafeng; Dornevil, Kednerlin; Wang, Siming; Zhang, Yong; Hendrich, Michael P.; Liu, Aimin

    2011-01-01

    An intriguing mystery about tryptophan 2,3-dioxygenase is its hydrogen peroxide-triggered enzyme reactivation from the resting ferric oxidation state to the catalytically active ferrous form. In this study, we found that such an odd Fe(III) reduction by an oxidant depends on the presence of l-Trp, which ultimately serves as the reductant for the enzyme. In the peroxide reaction with tryptophan 2,3-dioxygenase, a previously unknown catalase-like activity was detected. A ferryl species (δ = 0.055 mm/s and ΔEQ = 1.755 mm/s) and a protein-based free radical (g = 2.0028 and 1.72 millitesla linewidth) were characterized by Mössbauer and EPR spectroscopy, respectively. This is the first compound ES-type of ferryl intermediate from a heme-based dioxygenase characterized by EPR and Mössbauer spectroscopy. Density functional theory calculations revealed the contribution of secondary ligand sphere to the spectroscopic properties of the ferryl species. In the presence of l-Trp, the reactivation was demonstrated by enzyme assays and by various spectroscopic techniques. A Trp-Trp dimer and a monooxygenated l-Trp were both observed as the enzyme reactivation by-products by mass spectrometry. Together, these results lead to the unraveling of an over 60-year old mystery of peroxide reactivation mechanism. These results may shed light on how a metalloenzyme maintains its catalytic activity in an oxidizing environment. PMID:21632548

  18. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs

    PubMed Central

    Driggers, Camden M; Hartman, Steven J; Karplus, P Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ∼15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases. PMID:25307852

  19. Purification and characterization of hydroquinone dioxygenase from Sphingomonas sp. strain TTNP3

    PubMed Central

    2011-01-01

    Hydroquinone-1,2-dioxygenase, an enzyme involved in the degradation of alkylphenols in Sphingomonas sp. strain TTNP3 was purified to apparent homogeneity. The extradiol dioxygenase catalyzed the ring fission of hydroquinone to 4-hydroxymuconic semialdehyde and the degradation of chlorinated and several alkylated hydroquinones. The activity of 1 mg of the purified enzyme with unsubstituted hydroquinone was 6.1 μmol per minute, the apparent Km 2.2 μM. ICP-MS analysis revealed an iron content of 1.4 moles per mole enzyme. The enzyme lost activity upon exposure to oxygen, but could be reactivated by Fe(II) in presence of ascorbate. SDS-PAGE analysis of the purified enzyme yielded two bands of an apparent size of 38 kDa and 19 kDa, respectively. Data from MALDI-TOF analyses of peptides of the respective bands matched with the deduced amino acid sequences of two neighboring open reading frames found in genomic DNA of Sphingomonas sp strain TTNP3. The deduced amino acid sequences showed 62% and 47% identity to the large and small subunit of hydroquinone dioxygenase from Pseudomonas fluorescens strain ACB, respectively. This heterotetrameric enzyme is the first of its kind found in a strain of the genus Sphingomonas sensu latu. PMID:21906340

  20. Direct Ring Fission of Salicylate by a Salicylate 1,2-Dioxygenase Activity from Pseudaminobacter salicylatoxidans

    PubMed Central

    Hintner, Jan-Peter; Lechner, Christa; Riegert, Ulrich; Kuhm, Andrea Elisabeth; Storm, Thomas; Reemtsma, Thorsten; Stolz, Andreas

    2001-01-01

    In cell extracts of Pseudaminobacter salicylatoxidans strain BN12, an enzymatic activity was detected which converted salicylate in an oxygen-dependent but NAD(P)H-independent reaction to a product with an absorbance maximum at 283 nm. This metabolite was isolated, purified, and identified by mass spectrometry and 1H and 13C nuclear magnetic resonance spectroscopy as 2-oxohepta-3,5-dienedioic acid. This metabolite could be formed only by direct ring fission of salicylate by a 1,2-dioxygenase reaction. Cell extracts from P. salicylatoxidans also oxidized 5-aminosalicylate, 3-, 4-, and 5-chlorosalicylate, 3-, 4-, and 5-methylsalicylate, 3- and 5-hydroxysalicylate (gentisate), and 1-hydroxy-2-naphthoate. The dioxygenase was purified and shown to consist of four identical subunits with a molecular weight of about 45,000. The purified enzyme showed higher catalytic constants with gentisate or 1-hydroxy-2-naphthoate than with salicylate. It was therefore concluded that P. salicylatoxidans synthesized a gentisate 1,2-dioxygenase with an extraordinary substrate range, which also allowed the oxidation of salicylate. PMID:11698383

  1. Vanadium-based, extended catalytic lifetime catechol dioxygenases: evidence for a common catalyst.

    PubMed

    Yin, Cindy-Xing; Finke, Richard G

    2005-06-29

    In 1999, a catechol dioxygenase derived from a V-polyoxometalate was reported which was able to perform a record >100 000 total turnovers of 3,5-di-tert-butylcatechol oxygenation using O2 as the oxidant (Weiner, H.; Finke, R. G. J. Am. Chem. Soc. 1999, 121, 9831). An important goal is to better understand this and other vanadium-based catechol dioxygenases. Scrutiny of 11 literature reports of vanadium-based catechol dioxygenases yielded the insight that they all proceed with closely similar selectivities. This, in turn, led to a "common catalyst hypothesis" for the broad range of vanadium based catechol dioxygenase precatalysts presently known. The following three classes of V-based compounds, 10 complexes total, have been explored to test the common catalyst hypothesis: (i) six vanadium-based polyoxometalate precatalysts, (n-Bu4N)4H5PV14O42, (n-Bu4N)7SiW9V3O40, (n-Bu4N)5[(CH3CN)(x)Fe(II).SiW9V3O40], (n-Bu4N)9P2W15V3O62, (n-Bu4N)5Na2[(CH3CN)(x)Fe(II).P2W15V3O62], and (n-Bu4N)4H2-gamma-SiW10V2O40; (ii) three vanadium catecholate complexes, [V(V)O(DBSQ)(DTBC)]2, [Et3NH]2[V(IV)O(DBTC)2].2CH3OH, and [Na(CH3OH)2]2[V(V)(DTBC)3]2.4CH3OH (where DBSQ = 3,5-di-tert-butylsemiquinone anion and DTBC = 3,5-di-tert-butylcatecholate dianion), and (iii) simple VO(acac)2. Product selectivity studies, catalytic lifetime tests, electron paramagnetic resonance spectroscopy (EPR), negative ion mode electrospray ionization-mass spectrometry (negative ion ESI-MS), and kinetic studies provided compelling evidence for a common catalyst or catalyst resting state, namely, Pierpont's structurally characterized vanadyl semiquinone catecholate dimer complex, [VO(DBSQ)(DTBC)]2, formed from V-leaching from the precatalysts. The results provide a considerable simplification and unification of a previously disparate literature of V-based catechol dioxygenases.

  2. Identification of the dioxygenase-generated intermediate formed during biosynthesis of the dihydropyrrole moiety common to anthramycin and sibiromycin

    PubMed Central

    Saha, Shalini; Li, Wei; Gerratana, Barbara; Rokita, Steven E.

    2015-01-01

    A description of pyrrolo[1,4]benzodiazepine (PBD) biosynthesis is a prerequisite for engineering production of analogs with enhanced antitumor activity. Predicted dioxygenases Orf12 and SibV associated with dihydropyrrole biosynthesis in PBDs anthramycin and sibiromycin, respectively, were expressed and purified for activity studies. UV-visible spectroscopy revealed that these enzymes catalyze the regiospecific 2,3-extradiol dioxygenation of L-3,4-dihydroxyphenylalanine (L-DOPA) to form L-2,3-secodopa (λmax = 368 nm). 1H NMR spectroscopy indicates that L-2,3-secodopa cyclizes into the α-keto acid tautomer of L-4-(2-oxo-3-butenoic-acid)-4,5-dihydropyrrole-2-carboxylic acid (λmax = 414 nm). Thus, the dioxygenases are key for establishing the scaffold of the dihydropyrrole moiety. Kinetic studies suggest the dioxygenase product is relatively labile and is likely consumed rapidly by subsequent biosynthetic steps. The enzymatic product and dimeric state of these dioxygenases are conserved in dioxygenases involved in dihydropyrrole or pyrrolidine biosynthesis within both PBD and non-PBD pathways. PMID:25564379

  3. Oxidation of chlorinated olefins by Escherichia coli transformed with dimethyl sulfide monooxygenase genes or cumene dioxygenase genes.

    PubMed

    Takami, Wako; Yoshida, Takako; Nojiri, Hideaki; Yamane, Hisakazu; Omori, Toshio

    1999-04-01

    In the present work, it was shown that the dimethyl sulfide (DMS) monooxygenase and the cumene dioxygenase catalyzed oxidation of various chlorinated ethenes, propenes, and butenes. The specific activities of these oxygenases were determined for C(2) to C(4) chlorinated olefins, and the oxidation rates ranged from 0.19 to 4.18 nmol.min(-1).mg(-1) of dry cells by the DMS monooxygenase and from 0.19 to 1.29 nmol.min(-1).mg(-1) of dry cells by the cumene dioxygenase. The oxidation products were identified by gas chromatography-mass spectrometry. Most chlorinated olefins were monooxygenated by the DMS monooxygenase to yield chlorinated epoxides. In the case of the cumene dioxygenase, the substrates lacking any chlorine atom on double-bond carbon atoms were dioxygenated, and those with chlorine atoms attaching to double-bond carbon atoms were monooxygenated to yield allyl alcohols.

  4. SPECTROSCOPIC STUDIES OF THE ANAEROBIC ENZYME-SUBSTRATE COMPLEX OF CATECHOL 1,2-DIOXYGENASE

    PubMed Central

    Horsman, Geoff P.; Jirasek, Andrew; Vaillancourt, Frédéric H.; Barbosa, Christopher J.; Jarzecki, Andrzej A.; Xu, Changliang; Mekmouche, Yasmina; Spiro, Thomas G.; Lipscomb, John D.; Blades, Michael W.; Turner, Robin F.B.; Eltis, Lindsay D.

    2008-01-01

    The basis of the respective regiospecificities of intradiol and extradiol dioxygenase is poorly understood and may be linked to the protonation state of the bidentate-bound catechol in the enzyme:substrate complex. Previous ultraviolet resonance Raman (UVRR) and UV-visible (UV-vis) difference spectroscopic studies demonstrated that in extradiol dioxygenases, the catechol is bound to the Fe(II) as a monoanion. In this study, we use the same approaches to demonstrate that in catechol 1,2-dioxygenase (C12O), an intradiol enzyme, the catechol binds to the Fe(III) as a dianion. Specifically, features at 290 nm and 1550 cm−1 in the UV-vis and UVRR difference spectra, respectively, are assigned to dianionic catechol based on spectra of the model compound, ferric tris(catecholate). The UVRR spectroscopic band assignments are corroborated by density functional theory (DFT) calculations. In addition, negative features at 240 nm in UV-vis difference spectra and at 1600, 1210, and 1175 cm−1 in UVRR difference spectra match those of a tyrosinate model compound, consistent with protonation of the axial tyrosinate ligand when it is displaced from the ferric ion coordination sphere upon substrate binding. The DFT calculations ascribe the asymmetry of the bound dianionic substrate to the trans donor effect of an equatorially ligated tyrosinate ligand. In addition, the computations suggest that trans donation from the tyrosinate ligand may facilitate charge-transfer from the substrate to yield the iron-bound semiquinone transition state, which is capable of reacting with dioxygen. In illustrating the importance of ligand trans effects in a biological system, the current study demonstrates the power of combining difference UVRR and optical spectroscopies to probe metal ligation in solution. PMID:16316234

  5. Mechanism for catechol ring cleavage by non-heme iron intradiol dioxygenases: a hybrid DFT study.

    PubMed

    Borowski, Tomasz; Siegbahn, Per E M

    2006-10-04

    The mechanism of the catalytic reaction of protocatechuate 3,4-dioxygenase (3,4-PCD), a representative intradiol dioxygenase, was studied with the hybrid density functional method B3LYP. First, a smaller model involving only the iron first-shell ligands (His460, His462, and Tyr408) and the substrates (catechol and dioxygen) was used to probe various a priori plausible reaction mechanisms. Then, an extended model involving also the most important second-shell groups (Arg457, Gln477, and Tyr479) was used for the refinement of the preselected mechanisms. The computational results suggest that the chemical reactions constituting the catalytic cycle of intradiol dioxygenases involve: (1) binding of the substrate as a dianion, in agreement with experimental suggestions, (2) binding of dioxygen to the metal aided by an electron transfer from the substrate to O(2), (3) formation of a bridging peroxo intermediate and its conformational change, which opens the coordination site trans to His462, (4) binding of a neutral XOH ligand (H(2)O or Tyr447) at the open site, (5) proton transfer from XOH to the neighboring peroxo ligand yielding the hydroperoxo intermediate, (6) a Criegee rearrangement leading to the anhydride intermediate, and (7) hydrolysis of the anhydride to the final acyclic product. One of the most important results obtained is that the Criegee mechanism requires an in-plane orientation of the four atoms (two oxygen and two carbon atoms) mainly involved in the reaction. This orientation yields a good overlap between the two sigma orbitals involved, C-C sigma and O-O sigma, allowing an efficient electron flow between them. Another interesting result is that under some conditions, a homolytic O-O bond cleavage might compete with the Criegee rearrangement. The role of the second-shell residues and the substituent effects are also discussed.

  6. Control of Substrate Specificity by Active-Site Residues in Nitrobenzene Dioxygenase

    PubMed Central

    Ju, Kou-San; Parales, Rebecca E.

    2006-01-01

    Nitrobenzene 1,2-dioxygenase from Comamonas sp. strain JS765 catalyzes the initial reaction in nitrobenzene degradation, forming catechol and nitrite. The enzyme also oxidizes the aromatic rings of mono- and dinitrotoluenes at the nitro-substituted carbon, but the basis for this specificity is not understood. In this study, site-directed mutagenesis was used to modify the active site of nitrobenzene dioxygenase, and the contribution of specific residues in controlling substrate specificity and enzyme performance was evaluated. The activities of six mutant enzymes indicated that the residues at positions 258, 293, and 350 in the α subunit are important for determining regiospecificity with nitroarene substrates and enantiospecificity with naphthalene. The results provide an explanation for the characteristic specificity with nitroarene substrates. Based on the structure of nitrobenzene dioxygenase, substitution of valine for the asparagine at position 258 should eliminate a hydrogen bond between the substrate nitro group and the amino group of asparagine. Up to 99% of the mononitrotoluene oxidation products formed by the N258V mutant were nitrobenzyl alcohols rather than catechols, supporting the importance of this hydrogen bond in positioning substrates in the active site for ring oxidation. Similar results were obtained with an I350F mutant, where the formation of the hydrogen bond appeared to be prevented by steric interference. The specificity of enzymes with substitutions at position 293 varied depending on the residue present. Compared to the wild type, the F293Q mutant was 2.5 times faster at oxidizing 2,6-dinitrotoluene while retaining a similar Km for the substrate based on product formation rates and whole-cell kinetics. PMID:16517627

  7. The Crystal Structure of the Ring-Hydroxylating Dioxygenase from Sphingomonas CHY-1

    SciTech Connect

    Jakoncic,J.; Jouanneau, Y.; Meyer, C.; Stojanoff, V.

    2007-01-01

    The ring-hydroxylating dioxygenase (RHD) from Sphingomonas CHY-1 is remarkable due to its ability to initiate the oxidation of a wide range of polycyclic aromatic hydrocarbons (PAHs), including PAHs containing four- and five-fused rings, known pollutants for their toxic nature. Although the terminal oxygenase from CHY-1 exhibits limited sequence similarity with well characterized RHDs from the naphthalene dioxygenase family, the crystal structure determined to 1.85 {angstrom} by molecular replacement revealed the enzyme to share the same global {alpha}{sub 3}{beta}{sub 3} structural pattern. The catalytic domain distinguishes itself from other bacterial non-heme Rieske iron oxygenases by a substantially larger hydrophobic substrate binding pocket, the largest ever reported for this type of enzyme. While residues in the proximal region close to the mononuclear iron atom are conserved, the central region of the catalytic pocket is shaped mainly by the side chains of three amino acids, Phe350, Phe404 and Leu356, which contribute to the rather uniform trapezoidal shape of the pocket. Two flexible loops, LI and LII, exposed to the solvent seem to control the substrate access to the catalytic pocket and control the pocket length. Compared with other naphthalene dioxygenases residues Leu223 and Leu226, on loop LI, are moved towards the solvent, thus elongating the catalytic pocket by at least 2 {angstrom}. An 11 {angstrom} long water channel extends from the interface between the {alpha} and {beta} subunits to the catalytic site. The comparison of these structures with other known oxygenases suggests that the broad substrate specificity presented by the CHY-1 oxygenase is primarily due to the large size and particular topology of its catalytic pocket and provided the basis for the study of its reaction mechanism.

  8. Structure of the 2,4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP

    PubMed Central

    Keegan, R.; Lebedev, A.; Erskine, P.; Guo, J.; Wood, S. P.; Hopper, D. J.; Rigby, S. E. J.; Cooper, J. B.

    2014-01-01

    The enzyme 2,4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in the predominantly hydrophobic active-site pocket where it undergoes peroxide radical-mediated heterolysis. PMID:25195757

  9. Chemical Components from Aloe and their Inhibition of Indoleamine 2, 3-dioxygenase

    PubMed Central

    Sun, Ya Nan; Li, Lin Ying; Li, Wei; Kang, Jong Seong; Hwang, Inkyu; Kim, Young Ho

    2017-01-01

    Background: In Korea, Aloe is routinely ingested as a traditional medicine or as a component of health beverages. Objective: To research the inhibition of indoleamine 2, 3-dioxygenase (IDO) activities of components from Aloe. Materials and Methods: the compounds were isolated by a combination of silica gel and YMC Rp-18 column chromatography, and their structures were identified by analysis of spectroscopic data (1D, 2D-NMR, and MS). All of the isolated compounds were examined for their ability to inhibit IDO, which actively suppresses immune functions by catalyzing the rate limiting reaction in the conversion of tryptophan to kynurenine. Results: In this phytochemical study, 18 known compounds were isolated from aqueous dissolved Aloe exudates. All of the isolated compounds were examined for their ability to inhibit IDO activities for a series of anthraquinone derivatives (1-7) isolated from the Aloe extract; the IC50 values of these compounds ranged from 39.41 to 53.93 µM. Enzyme kinetic studies of their modes of inhibition indicated that all of the compounds were uncompetitive inhibitors. Conclusion: The aqueous dissolved Aloe exudate can be used as a source of novel natural IDO inhibitors and merit testing as therapeutic agents in the treatments of cancer and immunopathologic diseases, such as autoimmune, inflammatory, and allergic disorders. SUMMARY In this study, 18 known compounds were isolated from aqueous dissolved Aloe exudates. All of the isolated compounds were examined for their ability to inhibit indoleamine 2, 3-dioxygenase (IDO) activities for a series of anthraquinone derivatives (1−7) isolated from the Aloe extract. Abbreviation used: IDO: inhibit indoleamine 2, 3-dioxygenase, TMS: tetramethylsilane, HMQC: heteronuclear multiple quantum correlation, HMBC: heteronuclear multiple bond correlation, COSY: 1H-1H correlation spectroscopy, ESI-MS: Electrospray ionization mass spectrometry, DMSO: dimethyl sulfoxide PMID:28216884

  10. Structure of the 2, 4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP

    SciTech Connect

    Keegan, R.; Lebedev, A.; Erskine, P.; Guo, J.; Wood, S. P.; Hopper, D. J.; Rigby, S. E. J.; Cooper, J. B.

    2014-09-01

    The first X-ray structure of a 2, 4′-dihydroxyacetophenone dioxygenase from Alcaligenes sp. 4HAP at a resolution of 2.2 Å is reported. This structure establishes that the enzyme adopts the cupin-fold, forming compact dimers with a pronounced hydrophobic interface between the monomers. Each monomer possesses a catalytic ferrous iron that is coordinated by three histidines (76, 78 and 114) and an additional ligand which has been putatively assigned as a carbonate, although formate and acetate are possibilities. The enzyme 2, 4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2, 4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in

  11. Trichloroethylene removal and oxidation toxicity mediated by toluene dioxygenase of Pseudomonas putida

    SciTech Connect

    Heald, S.; Jenkins, R.O.

    1994-12-01

    Trichloroethylene (TCE) is a major ground water contaminant and potential health hazard in drinking water. This paper reports on the cometabolism of TCE by a wild-type strain of Pseudomonas putida containing an inducible toluene dioxygenase enzyme. The results show rapid TCE removal by the strain but severe oxidation toxicity and rapid cell death. This is also the first report of enhanced capacity of bacterial cells to remove TCE in the presence of dithiothreitol. Presented also is evidence for induction of toluene degradation by TCE. 17 refs., 2 figs., 2 tabs.

  12. Metabolism of Doubly para-Substituted Hydroxychlorobiphenyls by Bacterial Biphenyl Dioxygenases

    PubMed Central

    Pham, Thi Thanh My; Sondossi, Mohammad

    2015-01-01

    In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4′-dihydroxybiphenyl, 4-hydroxy-4′-chlorobiphenyl, 3-hydroxy-4,4′-dichlorobiphenyl, and 3,3′-dihydroxy-4,4′-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4′-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4′-chlorobiphenyl and 3,4-dihydroxy-4′-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4′-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4′-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3′-trihydroxy-4′-chlorobiphenyl with concomitant dechlorination, and 2,3,3′-trihydroxy-4′-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3′-Dihydroxy-4,4′-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3′-trihydroxy-4,4′-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear

  13. The reduction of the Rieske iron-sulfur cluster in naphthalene dioxygenase by X-rays.

    PubMed

    Karlsson, A; Parales, J V; Parales, R E; Gibson, D T; Eklund, H; Ramaswamy, S

    2000-01-15

    Naphthalene 1,2 dioxygenase (NDO) displays characteristic UV-Vis spectra depending on the oxidation state of the Rieske center. Investigations on crystals of NDO grown for X-ray diffraction experiments showed spectra characteristic of the oxidized form. Crystals reduced in an anaerobic glovebox using sodium-dithionite showed a characteristic reduced spectrum. Spectra of crystals (cooled to 100 K) after being exposed to X-rays for data collection showed spectra corresponding to a reduced Rieske iron center, demonstrating the ability of X-rays to change the oxidation state of the Rieske iron-sulfur cluster in NDO.

  14. Compound-Specific Isotope Analysis of Nitroaromatic Contaminant Transformations by Nitroarene Dioxygenases

    NASA Astrophysics Data System (ADS)

    Pati, Sarah G.; Kohler, Hans-Peter E.; Hofstetter, Thomas B.

    2014-05-01

    Dioxygenation is an important biochemical reaction that often initiates the mineralization of recalcitrant organic contaminants such as nitroaromatic explosives, chlorinated benzenes, and polycyclic aromatic hydrocarbons. However, to assess the extent of dioxygenation in contaminated environments is difficult because of competing transformation processes and further reactions of the dioxygenation products. Compound-specific isotope analysis (CSIA) offers a new approach to reliably quantify biodegradation initiated by dioxygenation based on changes in stable isotope ratios of the pollutant. For CSIA it is essential to know the kinetic isotope effects (KIEs) pertinent to the dioxygenation mechanism of organic contaminants. Unfortunately, the range of KIEs of such reactions is poorly constrained although many dioxygenase enzymes with a broad substrate specificity have been reported. Dioxygenase enzymes usually exhibit complex reaction kinetics involving multiple substrates and substrate-specific binding modes which makes the determination of KIEs challenging. The goal of this study was to explore the magnitude and variability of 13C-, 2H-, and 15N-KIEs for the dioxygenation of one contaminant class, that is nitroaromatic contaminants (NACs). To this end, we investigated the C, H, and N isotope fractionation during the dioxygenation of nitrobenzene (NB), 2-nitrotoluene (2-NT), and 3-nitrotoluene (3-NT) by pure cultures, E. coli clones, cell extracts, and purified enzymes. From isotope fractionations measured in the substrates and reaction products, we determined dioxygenation KIEs for different combinations of the three substrates with nitrobenzene dioxygenase (NBDO) and 2-nitrotoluene dioxygenase (2NTDO). The 13C-, 2H-, and 15N-KIEs for the dioxygenation of NB by NBDO were consistent for all experimental systems considered (i.e., Comamonas sp. Strain JS765, E. coli clones, cell extracts of E. coli clones, and purified NBDO). This observation suggests that the isotope

  15. [Indoleamine 2,3-Dioxygenase Activity during Fulvestrant Therapy for Multiple Metastatic Breast Cancer Patients].

    PubMed

    Sakurai, Kenichi; Fujisaki, Shigeru; Adachi, Keita; Suzuki, Shuhei; Masuo, Yuki; Nagashima, Saki; Hara, Yukiko; Hirano, Tomohiro; Enomoto, Katsuhisa; Tomita, Ryouichi; Gonda, Kenji

    2016-10-01

    We evaluated the clinical significance of indoleamine 2,3-dioxygenase(IDO)during fulvestrant therapyfor multiple metastatic breast cancer patients. IDO activitycan be measured using the tryptophan(Trp)/kynurenine(Kyn)ratio. Trp and Kyn were measured using high-performance liquid chromatography(HPLC). The serum Trp/Kyn level in patients with multiple metastatic breast cancer was lower than in patients without metastases. IDO activityincreased after breast cancer metastases developed. IDO activitywas correlated with the number of metastatic lesions during toremifene and fulvestrant therapy. These results suggested that measurement of the Trp/Kyn ratio is useful to evaluate immunological metastatic status during endocrine therapy.

  16. Heterologous Expression and Characterization of Two 1-Hydroxy-2-Naphthoic Acid Dioxygenases from Arthrobacter phenanthrenivorans

    PubMed Central

    Kavakiotis, Konstantinos; Kallimanis, Aristeidis; Kyrpides, Nikos C.; Drainas, Constantin; Koukkou, Anna-Irini

    2012-01-01

    A protein fraction exhibiting 1-hydroxy-2-naphthoic acid (1-H2NA) dioxygenase activity was purified via ion exchange, hydrophobic interactions, and gel filtration chromatography from Arthrobacter phenanthrenivorans sp. nov. strain Sphe3 isolated from a Greek creosote-oil-polluted site. Matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and tandem MS (MS-MS) analysis revealed that the amino acid sequences of oligopeptides of the major 45-kDa protein species, as analyzed by SDS-PAGE and silver staining, comprising 29% of the whole sequence, exhibited strong homology with 1-H2NA dioxygenase of Nocardioides sp. strain KP7. A BLAST search of the recently sequenced Sphe3 genome revealed two putative open reading frames, named diox1 and diox2, showing 90% nucleotide identity to each other and 85% identity at the amino acid level with the Nocardia sp. homologue. diox1 was found on an indigenous Sphe3 plasmid, whereas diox2 was located on the chromosome. Both genes were induced by the presence of phenanthrene used as a sole carbon and energy source, and as expected, both were subject to carbon catabolite repression. The relative RNA transcription level of the chromosomal (diox2) gene was significantly higher than that of its plasmid (diox1) homologue. Both diox1 and diox2 putative genes were PCR amplified, cloned, and overexpressed in Escherichia coli. Recombinant E. coli cells expressed 1-H2NA dioxygenase activity. Recombinant enzymes exhibited Michaelis-Menten kinetics with an apparent Km of 35 μM for Diox1 and 29 μM for Diox2, whereas they showed similar kinetic turnover characteristics with Kcat/Km values of 11 × 106 M−1 s−1 and 12 × 106 M−1 s−1, respectively. Occurrence of two diox1 and diox2 homologues in the Sphe3 genome implies that a replicative transposition event has contributed to the evolution of 1-H2NA dioxygenase in A. phenanthrenivorans. PMID:22101055

  17. Purification and characterization of gentisate 1,2-dioxygenases from Pseudomonas alcaligenes NCIB 9867 and Pseudomonas putida NCIB 9869.

    PubMed

    Feng, Y; Khoo, H E; Poh, C L

    1999-03-01

    Two 3-hydroxybenzoate-inducible gentisate 1,2-dioxygenases were purified to homogeneity from Pseudomonas alcaligenes NCIB 9867 (P25X) and Pseudomonas putida NCIB 9869 (P35X), respectively. The estimated molecular mass of the purified P25X gentisate 1, 2-dioxygenase was 154 kDa, with a subunit mass of 39 kDa. Its structure is deduced to be a tetramer. The pI of this enzyme was established to be 4.8 to 5.0. The subunit mass of P35X gentisate 1, 2-dioxygenase was 41 kDa, and this enzyme was deduced to exist as a dimer, with a native molecular mass of about 82 kDa. The pI of P35X gentisate 1,2-dioxygenase was around 4.6 to 4.8. Both of the gentisate 1,2-dioxygenases exhibited typical saturation kinetics and had apparent Kms of 92 and 143 microM for gentisate, respectively. Broad substrate specificities were exhibited towards alkyl and halogenated gentisate analogs. Both enzymes had similar kinetic turnover characteristics for gentisate, with kcat/Km values of 44.08 x 10(4) s-1 M-1 for the P25X enzyme and 39.34 x 10(4) s-1 M-1 for the P35X enzyme. Higher kcat/Km values were expressed by both enzymes against the substituted gentisates. Significant differences were observed between the N-terminal sequences of the first 23 amino acid residues of the P25X and P35X gentisate 1,2-dioxygenases. The P25X gentisate 1,2-dioxygenase was stable between pH 5.0 and 7.5, with the optimal pH around 8.0. The P35X enzyme showed a pH stability range between 7.0 and 9.0, and the optimum pH was also 8.0. The optimal temperature for both P25X and P35X gentisate 1, 2-dioxygenases was around 50 degrees C, but the P35X enzyme was more heat stable than that from P25X. Both enzymes were strongly stimulated by 0.1 mM Fe2+ but were completely inhibited by the presence of 5 mM Cu2+. Partial inhibition of both enzymes was also observed with 5 mM Mn2+, Zn2+, and EDTA.

  18. Catechol 1,2-dioxygenase from Acinetobacter calcoaceticus: purification and properties.

    PubMed Central

    Patel, R N; Hou, C T; Felix, A; Lillard, M O

    1976-01-01

    Procedures for the purification of catechol 1,2-dioxygenase from extracts of Acinetobacter calcoaceticus strain ADP-96 are described. The purified enzyme was homogeneous as judged by ultracentrifugation and acrylamide gel electrophoresis. The enzyme contained 2 g-atoms of iron per mol of protein. The enzyme had a broad substrate specificity and catalyzed the oxidation of catechol, 4-methylcatechol, 3-methylcatechol, and 3-isopropyl catechol. The activity of the enzyme was inhibited by heavy metals, sulfhydryl inhibitors, and substrate analogues. The molecular weight of the enzyme was 85,000 as estimated by filtration on Bio-Gel agarose and 81,000 as estimated by sedimentation equilibrium analysis. The subunit size determined by sodium dodecyl sulfate-gel electrophoresis was 40,000. The amino terminal amino acid was methionine. The amino acid composition and spectral properties of 1,2-dioxygenase are also presented. Antisera prepared against the purified enzyme cross-reacted and inhibited enzyme activity in crude extracts from the other strain of A. calcoaceticus, but failed to cross-react and inhibit isofunctional enzyme from organisms of the genera Pseudomonas, Alcaligenes, and Nocardia. Images PMID:58860

  19. Bioconversion of car-3-ene by a dioxygenase of Pleurotus sapidus.

    PubMed

    Lehnert, Nicole; Krings, Ulrich; Sydes, Daniel; Wittig, Maximilian; Berger, Ralf G

    2012-06-30

    Mycelium of the basidiomycete Pleurotus sapidus known to contain a novel dioxygenase was used for the bioconversion of car-3-ene [I]. After 4h of incubation 25.3mgL(-1) car-3-en-5-one [V], 5.4mgL(-1) car-3-en-2-one [VII], and 7.3mgL(-1) car-2-en-4-one [XV] accumulated as major oxidation products. The identity of the respective carenones and their corresponding alcohols was confirmed by comparison with MS and NMR spectral data obtained for synthesized authentic compounds. The peak areas of oxidation products were at least five times higher as compared with autoxidation. A radical mechanism similar to lipoxygenase catalysis was proposed and substantiated with detailed product analyses. The reduction of assumed car-3-ene hydroperoxides to the corresponding alcohols evidenced the radical initiated formation of hydroperoxides and confirmed the regio- and stereo-selectivity of the dioxygenase. The introduction of molecular oxygen into the bicyclic car-3-ene [I] molecule occurred at allylic positions of a cyclic isopentenyl moiety with a pronounced preference for the position adjacent to the non-substituted carbon atom of the C-C-double bond. This co-factor independent selective oxygenation presents an alternative to P450 mono-oxygenase based approaches for the production of terpene derived flavor compounds, pharmaceuticals and other fine chemicals.

  20. Structural Insight into the Dioxygenation of Nitroarene Compounds: the Crystal Structure of Nitrobenzene Dioxygenase

    SciTech Connect

    Friemann, Rosmarie; Ivkovic-Jensen, Maja M.; Lessner, Daniel J.; Yu, Chi-Li; Gibson, David T.; Parales, Rebecca E.; Eklund, Hans; Ramaswamy, S.

    2010-07-19

    Nitroaromatic compounds are used extensively in many industrial processes and have been released into the environment where they are considered environmental pollutants. Nitroaromatic compounds, in general, are resistant to oxidative attack due to the electron-withdrawing nature of the nitro groups and the stability of the benzene ring. However, the bacterium Comamonas sp. strain JS765 can grow with nitrobenzene as a sole source of carbon, nitrogen and energy. Biodegradation is initiated by the nitrobenzene dioxygenase (NBDO) system. We have determined the structure of NBDO, which has a hetero-hexameric structure similar to that of several other Rieske non-heme iron dioxygenases. The catalytic subunit contains a Rieske iron-sulfur center and an active-site mononuclear iron atom. The structures of complexes with substrates nitrobenzene and 3-nitrotoluene reveal the structural basis for its activity with nitroarenes. The substrate pocket contains an asparagine residue that forms a hydrogen bond to the nitro-group of the substrate, and orients the substrate in relation to the active-site mononuclear iron atom, positioning the molecule for oxidation at the nitro-substituted carbon.

  1. Oxidation of aminonitrotoluenes by 2,4-DNT dioxygenase of Burkholderia sp. strain DNT.

    PubMed

    Leungsakul, Thammajun; Keenan, Brendan G; Mori, Masa-aki; Morton, Martha D; Stuart, James D; Smets, Barth F; Wood, Thomas K

    2006-02-05

    Aminonitrotoluenes form rapidly from the reduction of dinitrotoluenes (DNTs) which are priority pollutants and animal carcinogens. For example, 4-amino-2-nitrotoluene (4A2NT) and 2A4NT accumulate from the reduction of 2,4-DNT during its aerobic biodegradation. Here, we show that 2,4-DNT dioxygenase (DDO) from Burkholderia sp. strain DNT oxidizes the aminonitrotoluenes 2A3NT, 2A6NT, 4A3NT, and 5A2NT to 2-amino-3-nitrobenzylalcohol, 2-amino-4-nitro-m-cresol and 3-amino-5-nitro-p-cresol, 4-amino-3-nitrobenzylalcohol and aminonitrocresol, and 2-amino-5-nitro-o-cresol, respectively. 2A5NT and 3A4NT are oxidized to aminonitrocresols and/or aminonitrobenzylalcohols, and 4A2NT is oxidized to aminonitrocresol. Only 2A4NT, a reduced compound derived from 2,4-DNT, was not oxidized by DDO or its three variants. The alpha subunit mutation I204Y resulted in two to fourfold faster oxidization of the aminonitrotoluenes. Though these enzymes are dioxygenases, they acted like monooxygenases by adding a single hydroxyl group, which did not result in the release of nitrite.

  2. A DFT Study of the cis-Dihydroxylation of Nitroaromatic Compounds Catalyzed by Nitrobenzene Dioxygenase

    PubMed Central

    2014-01-01

    The mechanism of cis-dihydroxylation of nitrobenzene and 2-nitrotoluene catalyzed by nitrobenzene 1,2-dioxygenase (NBDO), a member of the naphthalene family of Rieske non-heme iron dioxygenases, was studied by means of the density functional theory method using four models of the enzyme active site. Different possible reaction pathways for the substrate dioxygenation initiated either by the FeIII–OOH or HO–FeV=O attack on the aromatic ring were considered and the computed activation barriers compared with the Gibbs free energy of activation for the oxygen–oxygen cleavage leading to the formation of the iron(V)–oxo species from its ferric hydroperoxo precursor. The mechanism of the substrate cis-dihydroxylation leading to the formation of a cis-dihydrodiol was then investigated, and the most feasible mechanism was found to be starting with the attack of the high-valent iron–oxo species on the substrate ring yielding a radical intermediate, which further evolves toward the final product. PMID:24624972

  3. Molecular Characterization and Substrate Specificity of Nitrobenzene Dioxygenase from Comamonas sp. Strain JS765

    PubMed Central

    Lessner, Daniel J.; Johnson, Glenn R.; Parales, Rebecca E.; Spain, Jim C.; Gibson, David T.

    2002-01-01

    Comamonas sp. strain JS765 can grow with nitrobenzene as the sole source of carbon, nitrogen, and energy. We report here the sequence of the genes encoding nitrobenzene dioxygenase (NBDO), which catalyzes the first step in the degradation of nitrobenzene by strain JS765. The components of NBDO were designated ReductaseNBZ, FerredoxinNBZ, OxygenaseNBZα, and OxygenaseNBZβ, with the gene designations nbzAa, nbzAb, nbzAc, and nbzAd, respectively. Sequence analysis showed that the components of NBDO have a high level of homology with the naphthalene family of Rieske nonheme iron oxygenases, in particular, 2-nitrotoluene dioxygenase from Pseudomonas sp. strain JS42. The enzyme oxidizes a wide range of substrates, and relative reaction rates with partially purified OxygenaseNBZ revealed a preference for 3-nitrotoluene, which was shown to be a growth substrate for JS765. NBDO is the first member of the naphthalene family of Rieske nonheme iron oxygenases reported to oxidize all of the isomers of mono- and dinitrotoluenes with the concomitant release of nitrite. PMID:11823201

  4. A DFT study of the cis-dihydroxylation of nitroaromatic compounds catalyzed by nitrobenzene dioxygenase.

    PubMed

    Pabis, Anna; Geronimo, Inacrist; Paneth, Piotr

    2014-03-27

    The mechanism of cis-dihydroxylation of nitrobenzene and 2-nitrotoluene catalyzed by nitrobenzene 1,2-dioxygenase (NBDO), a member of the naphthalene family of Rieske non-heme iron dioxygenases, was studied by means of the density functional theory method using four models of the enzyme active site. Different possible reaction pathways for the substrate dioxygenation initiated either by the Fe(III)-OOH or HO-Fe(V)═O attack on the aromatic ring were considered and the computed activation barriers compared with the Gibbs free energy of activation for the oxygen-oxygen cleavage leading to the formation of the iron(V)-oxo species from its ferric hydroperoxo precursor. The mechanism of the substrate cis-dihydroxylation leading to the formation of a cis-dihydrodiol was then investigated, and the most feasible mechanism was found to be starting with the attack of the high-valent iron-oxo species on the substrate ring yielding a radical intermediate, which further evolves toward the final product.

  5. Occurrence of two different forms of protocatechuate 3,4-dioxygenase in a Moraxella sp.

    PubMed Central

    Sterjiades, R; Pelmont, J

    1989-01-01

    Two alternative forms of protocatechuate 3,4-dioxygenase (PCase) have been purified from Moraxella sp. strain GU2, a bacterium that is able to grow on guaiacol or various other phenolic compounds as the sole source of carbon and energy. One of these forms (PCase-P) was induced by protocatechuate and had an apparent molecular weight of 220,000. The second form (PCase-G) was induced by guaiacol or other phenolic compounds, such as 2-ethoxyphenol or 4-hydroxybenzoate. It appeared to be smaller (Mr 158,000), and its turnover number was about double that of the former enzyme. Both dioxygenases had similar properties and were built from the association of equal amounts of nonidentical subunits, alpha and beta, which were estimated to have molecular weights of 29,500 and 25,500, respectively. The (alpha beta)3 and (alpha beta)4 structures were suggested for PCases G and P, respectively. On the basis of two-dimensional gel electrophoresis, the alpha and beta polypeptides of PCase-G differed from those of PCase-P. Amino acid analysis supported this conclusion. Both PCases, however, had several other properties in common. It is proposed that both isoenzymes were generated from different sets of alpha and beta subunits, and the significance of these data is discussed. Images PMID:2541659

  6. Molecular insights into substrate recognition and catalysis by tryptophan 2,3-dioxygenase.

    PubMed

    Forouhar, Farhad; Anderson, J L Ross; Mowat, Christopher G; Vorobiev, Sergey M; Hussain, Arif; Abashidze, Mariam; Bruckmann, Chiara; Thackray, Sarah J; Seetharaman, Jayaraman; Tucker, Todd; Xiao, Rong; Ma, Li-Chung; Zhao, Li; Acton, Thomas B; Montelione, Gaetano T; Chapman, Stephen K; Tong, Liang

    2007-01-09

    Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-A resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate L-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the L-stereospecificity. A second, possibly allosteric, L-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of L-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.

  7. Purification and properties of pyrazon dioxygenase from pyrazon-degrading bacteria.

    PubMed

    Sauber, K; Fröhner, C; Rosenberg, G; Eberspächer, J; Lingens, F

    1977-03-15

    Chromatography on DEAE-cellulose and gel filtration on Sephadex revealed that pyrazon dioxygenase from pyrazon-degrading bacteria consists of three different enzyme components. No component alone oxidizes the phenyl moiety of pyrazon, only when the three components are combined can oxidation be detected. Following electron paramagnetic resonance and ultraviolet measurements the protein nature of the three components was determined: component A1 (molecular weight about 180000,red-brown in colour) is an iron-sulphur protein. The existence of approximately two moles of iron and two moles of inorganic sulphur per mole of protein was demonstrated. This enzyme component was purified to homogeneity in disc electrophoresis. Component A2 is a yellow protein of a molecular weight of about 67000. FAD was shown to be the prosthetic group of this protein. Component B (molecular weight about 12000, brown in colour) is a protein of the ferredoxin type, which was purified to homogeneity, as demonstrated by disc electrophoresis. A hypothetical scheme for the cooperation of the three components is proposed: component A2 accepts as cosubstrate NADH and functions as a ferredoxin reductase. The ferredoxin, component B, has the function of an electron carrier. The conversion of the substrates is effected by component A1, the terminal dioxygenase.

  8. Molecular Insights into Substrate Recognition and Catalysis by Tryptophan 2,3-dioxygenase

    SciTech Connect

    Forouhar,F.; Ross Anderson, J.; Mowat, C.; Vorobiev, S.; Hussain, A.; Abashidze, M.; Bruckmann, C.; Thackray, S.; Seetharaman, J.; et al.

    2007-01-01

    Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) constitute an important, yet relatively poorly understood, family of heme-containing enzymes. Here, we report extensive structural and biochemical studies of the Xanthomonas campestris TDO and a related protein SO4414 from Shewanella oneidensis, including the structure at 1.6-{angstrom} resolution of the catalytically active, ferrous form of TDO in a binary complex with the substrate l-Trp. The carboxylate and ammonium moieties of tryptophan are recognized by electrostatic and hydrogen-bonding interactions with the enzyme and a propionate group of the heme, thus defining the l-stereospecificity. A second, possibly allosteric, l-Trp-binding site is present at the tetramer interface. The sixth coordination site of the heme-iron is vacant, providing a dioxygen-binding site that would also involve interactions with the ammonium moiety of l-Trp and the amide nitrogen of a glycine residue. The indole ring is positioned correctly for oxygenation at the C2 and C3 atoms. The active site is fully formed only in the binary complex, and biochemical experiments confirm this induced-fit behavior of the enzyme. The active site is completely devoid of water during catalysis, which is supported by our electrochemical studies showing significant stabilization of the enzyme upon substrate binding.

  9. Isolation and characterization of a novel strain of Stenotrophomonas maltophilia possessing various dioxygenases for monocyclic hydrocarbon degradation

    PubMed Central

    Urszula, Guzik; Izabela, Greń; Danuta, Wojcieszyńska; Sylwia, Łabużek

    2009-01-01

    A Gram-negative bacterium, designated as strain KB2, was isolated from activated sludge and was found to utilize different aromatic substrates as sole carbon and energy source. On the basis of morphological and physiochemical characteristics and 16S rRNA gene sequence analysis, the isolated strain KB2 was identified as Stenotrophomonas maltophilia. Strain KB2 is from among different Stenotrophomonas maltophilia strains the first one described as exhibiting the activities of three types of dioxygenases depending on the structure of the inducer. The cells grown on benzoate and catechol showed mainly catechol 1,2-dioxygenase activity. The activity of 2,3-dioxygenase was detected after phenol induction. Protocatechuate 3,4-dioxygenase was found in crude cell extracts of this strain after incubation with 4-hydroxybenzoic acid, protocatechuic acid and vanillic acid. Because of broad spectrum of dioxygenases’ types that Stenotrophomonas maltophilia KB2 can exhibit, this strain appears to be very powerful and useful tool in the biotreatment of wastewaters and in soil decontamination. PMID:24031359

  10. Photosystem II-inhibitors play a limited role in sweet corn response to 4-hydroxyphenyl pyruvate dioxygenase-inhibiting herbicides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Postemergence (POST) application of 4-hydroxyphenyl pyruvate dioxygenase (HPPD) inhibitors in combination with a photosystem II (PSII) inhibitor, such as atrazine, is common practice in sweet corn production. Given the sensitivity of sweet corn to HPPD-inhibiting herbicides, the objective of this wo...

  11. An investigation of the iron-sulphur proteins of benzene dioxygenase from Pseudomonas putida by electron-spin-resonance spectroscopy.

    PubMed Central

    Geary, P J; Saboowalla, F; Patil, D; Cammack, R

    1984-01-01

    Benzene dioxygenase from Pseudomonas putida comprises three components, namely a flavoprotein (NADH:ferredoxin oxidoreductase; Mr 81000), an intermediate electron-transfer protein, or ferredoxin (Mr 12000) with a [2Fe-2S] cluster, and a terminal dioxygenase containing two [2Fe-2S] iron-sulphur clusters (Mr 215000), which requires two additional Fe2+ atoms/molecule for oxygenase activity. The ferredoxin and the dioxygenase give e.s.r. signals in the reduced state with rhombic symmetry and average g values of 1.92 and 1.896 respectively. The mid-point redox potentials were determined by e.s.r. titration at pH 7.0 to be -155 mV and -112 mV respectively. The signal from the dioxygenase shows pronounced g anisotropy and most closely resembles those of 4-methoxybenzoate mono-oxygenase from Pseudomonas putida and the [2Fe-2S] 'Rieske' proteins of the quinone-cytochrome c region of electron-transport chains of respiration and photosynthesis. PMID:6324743

  12. Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase.

    PubMed

    Iwakiri, Ryo; Yoshihira, Kunichika; Ngadiman; Futagami, Taiki; Goto, Masatoshi; Furukawa, Kensuke

    2004-06-01

    We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.

  13. Alcaligenes eutrophus JMP134 "2,4-dichlorophenoxyacetate monooxygenase" is an alpha-ketoglutarate-dependent dioxygenase.

    PubMed Central

    Fukumori, F; Hausinger, R P

    1993-01-01

    The Alcaligenes eutrophus JMP134 tfdA gene, encoding the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation, was overexpressed in Escherichia coli, and several enzymatic properties of the partially purified gene product were examined. Although the tfdA-encoded enzyme is typically referred to as 2,4-D monooxygenase, we were unable to observe any reductant-dependent activity. Rather, we demonstrate that this enzyme is a ferrous ion-dependent dioxygenase that uses alpha-ketoglutarate as a cosubstrate. The alpha-ketoglutarate is converted to succinate concomitant with 2,4-D conversion to 2,4-dichlorophenol. By using [1-14C]alpha-ketoglutarate, we established that carbon dioxide is the second product derived from alpha-ketoglutarate. Finally, we verified the proposal that glyoxylate is the second product derived from 2,4-D. PMID:8458850

  14. A fluorescence-based assay for indoleamine 2,3-dioxygenase.

    PubMed

    Matin, Azadeh; Streete, Isla M; Jamie, Ian M; Truscott, Roger J W; Jamie, Joanne F

    2006-02-01

    A rapid and sensitive fluorescence-based bioassay for determination of indoleamine 2,3-dioxygenase (IDO) activity has been developed. This assay relies on the quantification of the amount of kynurenine produced in the assay medium by fluorescence and complements the standard absorbance and high-performance liquid chromatography (HPLC) assay methods. The fluorescence method has limits of detection similar to those of the standard assay methods. Measured activities of IDO, including in the presence of tryptophan-based inhibitors, were in statistical agreement with the absorbance and HPLC assay methods. The fluorescence-based assay was also suitable for assessment of IDO inhibition by compounds that are incompatible with the absorbance method.

  15. Functional diversity of 2-oxoglutarate/Fe(II)-dependent dioxygenases in plant metabolism

    PubMed Central

    Farrow, Scott C.; Facchini, Peter J.

    2014-01-01

    Oxidative enzymes catalyze many different reactions in plant metabolism. Among this suite of enzymes are the 2-oxoglutarate/Fe(II)-dependent dioxygenases (2-ODDs). Cytochromes P450 (CYPs) as often considered the most versatile oxidative enzymes in nature, but the diversity and complexity of reactions catalyzed by 2-ODDs is superior to the CYPs. The list of oxidative reactions catalyzed by 2-ODDs includes hydroxylations, demethylations, desaturations, ring closure, ring cleavage, epimerization, rearrangement, halogenation, and demethylenation. Furthermore, recent work, including the discovery of 2-ODDs involved in epigenetic regulation, and others catalyzing several characteristic steps in specialized metabolic pathways, support the argument that 2-ODDs are among the most versatile and important oxidizing biological catalysts. In this review, we survey and summarize the pertinent literature with a focus on several key reactions catalyzed by 2-ODDs, and discuss the significance and impact of these enzymes in plant metabolism. PMID:25346740

  16. Modified CAROTENOID CLEAVAGE DIOXYGENASE8 expression correlates with altered branching in kiwifruit (Actinidia chinensis).

    PubMed

    Ledger, Susan E; Janssen, Bart J; Karunairetnam, Sakuntala; Wang, Tianchi; Snowden, Kimberley C

    2010-11-01

    • CAROTENOID CLEAVAGE DIOXYGENASE (CCD) genes have been demonstrated to play an integral role in the control of branch development in model plants, including Arabidopsis, pea (Pisum sativum), petunia (Petunia hybrida) and rice (Oryza sativa). • Actinidia chinensis is a woody perennial plant grown for commercial production of kiwifruit. CCD7 and CCD8 genes were isolated from A. chinensis and these genes are predominantly expressed in the roots of kiwifruit. AcCCD7 and AcCCD8 were able to complement the corresponding Arabidopsis mutants max3 and max4. The function of AcCCD8 in branch development was determined in transgenic kiwifruit plants containing an RNAi construct for AcCCD8. • Reduction in expression of AcCCD8 correlated with an increase in branch development and delayed leaf senescence. • The CCD pathway for control of branch development is conserved across a wide range of species, including kiwifruit, a woody perennial.

  17. 2-Oxoglutarate-dependent dioxygenases in the biosynthesis of simple coumarins

    PubMed Central

    Shimizu, Bun-Ichi

    2014-01-01

    Coumarins are natural plant products that have been the subject of extensive phytochemical and pharmacological research studies in the past few decades. The core structure of coumarins is derived from the respective cinnamates via ortho-hydroxylation of the aromatic ring, trans/cis isomerization, and lactonization. Various substitution patterns of coumarins have been reported, whereas the biosynthesis of coumarins remains elusive. Ortho-hydroxylation is a key step in simple coumarin biosynthesis as a branch point from the lignin biosynthetic pathway. 2-Oxoglutarate-dependent dioxygenases (2OGDs) from plants convert cinnamate derivatives into simple coumarins through the process of ortho-hydroxylation. This review describes the 2OGDs involved in coumarin biosynthesis and their substrate specificities. PMID:25404933

  18. Substrate-protein interaction in human tryptophan dioxygenase: the critical role of H76.

    PubMed

    Batabyal, Dipanwita; Yeh, Syun-Ru

    2009-03-11

    The initial and rate-limiting step of the kynurenine pathway in humans involves the oxidation of tryptophan to N-formyl kynurenine catalyzed by two hemeproteins, tryptophan 2,3-dioxygenase (hTDO) and indoleamine 2,3-dioxygenase (hIDO). In hTDO, the conserved H76 residue is believed to act as an active site base to deprotonate the indole NH group of Trp, the initial step of the Trp oxidation reaction. In hIDO, this histidine is replaced by a serine. To investigate the role of the H76, we have studied the H76S and H76A mutants of hTDO. Activity assays show that the mutations cause a decrease in k(cat) and an increase in K(M) for both mutants. The decrease in the k(cat) is accounted for by the replacement of the active site base catalyst, H76, with a weaker base, possibly a water, whereas the increase in K(M) is attributed to the loss of the specific interactions between the H76 and the substrate as well as the protein matrix. Resonance Raman studies with various Trp analogs indicate that the substrate is positioned in the active site by the ammonium, carboxylate, and indole groups, via intricate H-bonding and hydrophobic interactions. This scenario is consistent with the observation that l-Trp binding significantly perturbs the electronic properties of the O(2)-adduct of hTDO. The important structural and functional roles of H76 in hTDO is underscored by the observation that the electronic configuration of the active ternary complex, l-Trp-O(2)-hTDO, is sensitive to the H76 mutations.

  19. Molecular Pathways: Targeting IDO1 and Other Tryptophan Dioxygenases for Cancer Immunotherapy.

    PubMed

    Zhai, Lijie; Spranger, Stefani; Binder, David C; Gritsina, Galina; Lauing, Kristen L; Giles, Francis J; Wainwright, Derek A

    2015-12-15

    Indoleamine 2, 3-dioxygenase 1 (IDO1), IDO2, and tryptophan 2, 3-dioxygenase (TDO) comprise a family of enzymes that catalyze the first- and rate-limiting step associated with the catabolic conversion of tryptophan (Trp) into kynurenine (Kyn). Through subsequent enzymatic and spontaneous reactions, Kyn is further converted into the energetic substrates, NAD(+) and ATP, to fuel cellular metabolic functions. Coincidently, the depletion of Trp and accumulation of Kyn has been demonstrated to induce effector T-cell apoptosis/dysfunction and immunosuppressive regulatory T-cell induction, respectively. Similar to other immune checkpoints, IDO1 and TDO are suggested to be important targets for immunotherapeutic intervention. This is represented by the recent growth of efforts to inhibit the Trp-to-Kyn pathway as a means to control immunosuppression. Inhibitors currently in clinical trials, INCB024360, GDC-0919, indoximod, and an IDO1 peptide-based vaccine, are being evaluated for their efficacy against a wide range of cancers including melanoma, glioblastoma, non-small cell lung, pancreatic, and/or breast cancer, as well as metastatic disease. Despite the rapid development of potent clinical grade inhibitors, strategic questions remain. Here, we review the state of the literature with respect to current therapeutic inhibitors of tryptophan catabolism, evaluation of those efforts preclinically and clinically, compensatory changes that occur with therapeutic targeting, as well as newly recognized signaling features that raise critical questions to the field. Given the rapidly evolving interest in determining how IDO1/TDO, and to an unknown extent, IDO2, can be targeted for increasing cancer immunotherapeutic efficacy, we present a brief but comprehensive analysis that addresses critical questions, while highlighting the mechanics that remain to be explored.

  20. Functional Metagenomics of a Biostimulated Petroleum-Contaminated Soil Reveals an Extraordinary Diversity of Extradiol Dioxygenases.

    PubMed

    Terrón-González, Laura; Martín-Cabello, Guadalupe; Ferrer, Manuel; Santero, Eduardo

    2016-04-01

    A metagenomic library of a petroleum-contaminated soil was constructed in a fosmid vector that allowed heterologous expression of metagenomic DNA. The library, consisting of 6.5 Gb of metagenomic DNA, was screened for extradiol dioxygenase (Edo) activity using catechol and 2,3-dihydroxybiphenyl as the substrates. Fifty-eight independent clones encoding extradiol dioxygenase activity were identified. Forty-one different Edo-encoding genes were identified. The population of Edo genes was not dominated by a particular gene or by highly similar genes; rather, the genes had an even distribution and high diversity. Phylogenetic analyses revealed that most of the genes could not be ascribed to previously defined subfamilies of Edos. Rather, the Edo genes led to the definition of 10 new subfamilies of type I Edos. Phylogenetic analysis of type II enzymes defined 7 families, 2 of which harbored the type II Edos that were found in this work. Particularly striking was the diversity found in family I.3 Edos; 15 out of the 17 sequences assigned to this family belonged to 7 newly defined subfamilies. A strong bias was found that depended on the substrate used for the screening: catechol mainly led to the detection of Edos belonging to the I.2 family, while 2,3-dihydroxybiphenyl led to the detection of most other Edos. Members of the I.2 family showed a clear substrate preference for monocyclic substrates, while those from the I.3 family showed a broader substrate range and high activity toward 2,3-dihydroxybiphenyl. This metagenomic analysis has substantially increased our knowledge of the existing biodiversity of Edos.

  1. Promotion of Germination Using Hydroxamic Acid Inhibitors of 9-cis-Epoxycarotenoid Dioxygenase

    PubMed Central

    Awan, Sajjad Z.; Chandler, Jake O.; Harrison, Peter J.; Sergeant, Martin J.; Bugg, Timothy D. H.; Thompson, Andrew J.

    2017-01-01

    Abscisic acid (ABA) inhibits seed germination and the regulation of ABA biosynthesis has a role in maintenance of seed dormancy. The key rate-limiting step in ABA biosynthesis is catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED). Two hydroxamic acid inhibitors of carotenoid cleavage dioxygenase (CCD), D4 and D7, previously found to inhibit CCD and NCED in vitro, are shown to have the novel property of decreasing mean germination time of tomato (Solanum lycopersicum L.) seeds constitutively overexpressing LeNCED1. Post-germination, D4 exhibited no negative effects on tomato seedling growth in terms of height, dry weight, and fresh weight. Tobacco (Nicotiana tabacum L.) seeds containing a tetracycline-inducible LeNCED1 transgene were used to show that germination could be negatively and positively controlled through the chemical induction of gene expression and the chemical inhibition of the NCED protein: application of tetracycline increased mean germination time and delayed hypocotyl emergence in a similar manner to that observed when exogenous ABA was applied and this was reversed by D4 when NCED expression was induced at intermediate levels. D4 also improved germination in lettuce (Lactuca sativa L.) seeds under thermoinhibitory temperatures and in tomato seeds imbibed in high osmolarity solutions of polyethylene glycol. D4 reduced ABA and dihydrophaseic acid accumulation in tomato seeds overexpressing LeNCED1 and reduced ABA accumulation in wild type tomato seeds imbibed on polyethylene glycol. The evidence supports a mode of action of D4 through NCED inhibition, and this molecule provides a lead compound for the design of NCED inhibitors with greater specificity and potency. PMID:28373878

  2. Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase

    PubMed Central

    Kumar, Murugaeson R.; Zapata, Adrian; Ramirez, Alejandro J.; Bowen, Sara K.; Francisco, Wilson A.; Farmer, Patrick J.

    2011-01-01

    Quercetin dioxygenase (QDO) catalyzes the oxidation of the flavonol quercetin with dioxygen, cleaving the central heterocyclic ring and releasing CO. The QDO from Bacillus subtilis is unusual in that it has been shown to be active with several divalent metal cofactors such as Fe, Mn, and Co. Previous comparison of the catalytic activities suggest that Mn(II) is the preferred cofactor for this enzyme. We herein report the unprecedented substitution of nitrosyl hydride (HNO) for dioxygen in the activity of Mn-QDO, resulting in the incorporation of both N and O atoms into the product. Turnover is demonstrated by consumption of quercetin and other related substrates under anaerobic conditions in the presence of HNO-releasing compounds and the enzyme. As with dioxygenase activity, a nonenzymatic base-catalyzed reaction of quercetin with HNO is observed above pH 7, but no enhancement of this basal reactivity is found upon addition of divalent metal salts. Unique and regioselective N-containing products (14N/15N) have been characterized by MS analysis for both the enzymatic and nonenzymatic reactions. Of the several metallo-QDO enzymes examined for nitroxygenase activity under anaerobic condition, only the Mn(II) is active; the Fe(II) and Co(II) substituted enzymes show little or no activity. This result represents an enzymatic catalysis which we denote nitroxygenase activity; the unique reactivity of the Mn-QDO suggests a metal-mediated electron transfer mechanism rather than metal activation of the substrate’s inherent base-catalyzed reactivity. PMID:22084064

  3. Spliceostatin hemiketal biosynthesis in Burkholderia spp. is catalyzed by an iron/α-ketoglutarate–dependent dioxygenase

    PubMed Central

    Eustáquio, Alessandra S.; Janso, Jeffrey E.; Ratnayake, Anokha S.; O’Donnell, Christopher J.; Koehn, Frank E.

    2014-01-01

    Spliceostatins are potent spliceosome inhibitors biosynthesized by a hybrid nonribosomal peptide synthetase−polyketide synthase (NRPS−PKS) system of the trans-acyl transferase (AT) type. Burkholderia sp. FERM BP-3421 produces hemiketal spliceostatins, such as FR901464, as well as analogs containing a terminal carboxylic acid. We provide genetic and biochemical evidence for hemiketal biosynthesis by oxidative decarboxylation rather than the previously hypothesized Baeyer–Villiger oxidative release postulated to be catalyzed by a flavin-dependent monooxygenase (FMO) activity internal to the last module of the PKS. Inactivation of Fe(II)/α-ketoglutarate–dependent dioxygenase gene fr9P led to loss of hemiketal congeners, whereas the mutant was still able to produce all major carboxylic acid-type compounds. FMO mutants, on the other hand, produced both hemiketal and carboxylic acid analogs containing an exocyclic methylene instead of an epoxide, indicating that the FMO is involved in epoxidation rather than Baeyer–Villiger oxidation. Moreover, recombinant Fr9P enzyme was shown to catalyze hydroxylation to form β-hydroxy acids, which upon decarboxylation led to hemiketal FR901464. Finally, a third oxygenase activity encoded in the biosynthetic gene cluster, the cytochrome P450 monooxygenase Fr9R, was assigned as a 4-hydroxylase based on gene inactivation results. Identification and deletion of the gene involved in hemiketal formation allowed us to generate a strain—the dioxygenase fr9P− mutant—that accumulates only the carboxylic acid-type spliceostatins, which are as potent as the hemiketal analogs, when derivatized to increase cell permeability, but are chemically more stable. PMID:25097259

  4. Desipramine decreases expression of human and murine indoleamine-2,3-dioxygenases.

    PubMed

    Brooks, Alexandra K; Janda, Tiffany M; Lawson, Marcus A; Rytych, Jennifer L; Smith, Robin A; Ocampo-Solis, Cecilia; McCusker, Robert H

    2017-05-01

    Abundant evidence connects depression symptomology with immune system activation, stress and subsequently elevated levels of kynurenine. Anti-depressants, such as the tricyclic norepinephrine/serotonin reuptake inhibitor desipramine (Desip), were developed under the premise that increasing extracellular neurotransmitter level was the sole mechanism by which they alleviate depressive symptomologies. However, evidence suggests that anti-depressants have additional actions that contribute to their therapeutic potential. The Kynurenine Pathway produces tryptophan metabolites that modulate neurotransmitter activity. This recognition identified another putative pathway for anti-depressant targeting. Considering a recognized role of the Kynurenine Pathway in depression, we investigated the potential for Desip to alter expression of rate-limiting enzymes of this pathway: indoleamine-2,3-dioxygenases (Ido1 and Ido2). Mice were administered lipopolysaccharide (LPS) or synthetic glucocorticoid dexamethasone (Dex) with Desip to determine if Desip alters indoleamine-dioxygenase (DO) expression in vivo following a modeled immune and stress response. This work was followed by treating murine and human peripheral blood mononuclear cells (PBMCs) with interferon-gamma (IFNγ) and Desip. In vivo: Desip blocked LPS-induced Ido1 expression in hippocampi, astrocytes, microglia and PBMCs and Ido2 expression by PBMCs. Ex vivo: Desip decreased IFNγ-induced Ido1 and Ido2 expression in murine PBMCs. This effect was directly translatable to the human system as Desip decreased IDO1 and IDO2 expression by human PBMCs. These data demonstrate for the first time that an anti-depressant alters expression of Ido1 and Ido2, identifying a possible new mechanism behind anti-depressant activity. Furthermore, we propose the assessment of PBMCs for anti-depressant responsiveness using IDO expression as a biomarker.

  5. Functional Metagenomics of a Biostimulated Petroleum-Contaminated Soil Reveals an Extraordinary Diversity of Extradiol Dioxygenases

    PubMed Central

    Terrón-González, Laura; Martín-Cabello, Guadalupe; Ferrer, Manuel

    2016-01-01

    A metagenomic library of a petroleum-contaminated soil was constructed in a fosmid vector that allowed heterologous expression of metagenomic DNA. The library, consisting of 6.5 Gb of metagenomic DNA, was screened for extradiol dioxygenase (Edo) activity using catechol and 2,3-dihydroxybiphenyl as the substrates. Fifty-eight independent clones encoding extradiol dioxygenase activity were identified. Forty-one different Edo-encoding genes were identified. The population of Edo genes was not dominated by a particular gene or by highly similar genes; rather, the genes had an even distribution and high diversity. Phylogenetic analyses revealed that most of the genes could not be ascribed to previously defined subfamilies of Edos. Rather, the Edo genes led to the definition of 10 new subfamilies of type I Edos. Phylogenetic analysis of type II enzymes defined 7 families, 2 of which harbored the type II Edos that were found in this work. Particularly striking was the diversity found in family I.3 Edos; 15 out of the 17 sequences assigned to this family belonged to 7 newly defined subfamilies. A strong bias was found that depended on the substrate used for the screening: catechol mainly led to the detection of Edos belonging to the I.2 family, while 2,3-dihydroxybiphenyl led to the detection of most other Edos. Members of the I.2 family showed a clear substrate preference for monocyclic substrates, while those from the I.3 family showed a broader substrate range and high activity toward 2,3-dihydroxybiphenyl. This metagenomic analysis has substantially increased our knowledge of the existing biodiversity of Edos. PMID:26896130

  6. Crystallographic Comparison of Manganese- and Iron-Dependent Homoprotocatechuate 2,3-Dioxygenases

    PubMed Central

    Vetting, Matthew W.; Wackett, Lawrence P.; Que, Lawrence; Lipscomb, John D.; Ohlendorf, Douglas H.

    2004-01-01

    The X-ray crystal structures of homoprotocatechuate 2,3-dioxygenases isolated from Arthrobacter globiformis and Brevibacterium fuscum have been determined to high resolution. These enzymes exhibit 83% sequence identity, yet their activities depend on different transition metals, Mn2+ and Fe2+, respectively. The structures allow the origins of metal ion selectivity and aspects of the molecular mechanism to be examined in detail. The homotetrameric enzymes belong to the type I family of extradiol dioxygenases (vicinal oxygen chelate superfamily); each monomer has four βαβββ modules forming two structurally homologous N-terminal and C-terminal barrel-shaped domains. The active-site metal is located in the C-terminal barrel and is ligated by two equatorial ligands, H214NE1 and E267OE1; one axial ligand, H155NE1; and two to three water molecules. The first and second coordination spheres of these enzymes are virtually identical (root mean square difference over all atoms, 0.19 Å), suggesting that the metal selectivity must be due to changes at a significant distance from the metal and/or changes that occur during folding. The substrate (2,3-dihydroxyphenylacetate [HPCA]) chelates the metal asymmetrically at sites trans to the two imidazole ligands and interacts with a unique, mobile C-terminal loop. The loop closes over the bound substrate, presumably to seal the active site as the oxygen activation process commences. An “open” coordination site trans to E267 is the likely binding site for O2. The geometry of the enzyme-substrate complexes suggests that if a transiently formed metal-superoxide complex attacks the substrate without dissociation from the metal, it must do so at the C-3 position. Second-sphere active-site residues that are positioned to interact with the HPCA and/or bound O2 during catalysis are identified and discussed in the context of current mechanistic hypotheses. PMID:15028678

  7. Cis-2', 3'-dihydrodiol production on flavone B-ring by biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 expressed in Escherichia coli.

    PubMed

    Kim, Song-Young; Jung, Jihyun; Lim, Yoongho; Ahn, Joong-Hoon; Kim, Su-Il; Hur, Hor-Gil

    2003-01-01

    Escherichia coli JM109 strains expressing either toluene dioxygenase from Pseudomonas putida F1 or biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 were examined for their ability to catalyze flavones. Biphenyl dioxygenase produced metabolites from flavone and 5,7-dihydroxyflavone which were not found in the control experiments. The absorption maxima of UV-visible spectra for the metabolites from flavone and 5,7-dihydroxyflavone were found at 337 and 348 nm respectively by using a photodiode array detector in the HPLC. Liquid chromatography/mass spectroscopy (LC/MS) showed molecular weights 256 and 288 for the metabolites, respectively. The metabolite of flavone, which was isolated and purified from the bacterial culture, was further subjected to analysis by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. Based on the LC/MS and NMR results, biphenyl dioxygenase inserted oxygen at C2' and C3' on the B-ring of flavone, resulting in the formation of flavone cis-2', 3'-dihydrodiol (2-[3,4-dihydroxy-1.5-cyclohexadienyl]-4H-chromen-4-one). Since this product is not found in Chemical Abstracts, this compound is considered a novel one. In addition, biotransformation of flavones by biphenyl dioxygenase suggested a potential role of bacterial dioxygenase to synthesize novel compounds from plant secondary metabolites.

  8. Degradation of Chloroaromatics: Purification and Characterization of a Novel Type of Chlorocatechol 2,3-Dioxygenase of Pseudomonas putida GJ31

    PubMed Central

    Kaschabek, Stefan R.; Kasberg, Thomas; Müller, Dagmar; Mars, Astrid E.; Janssen, Dick B.; Reineke, Walter

    1998-01-01

    A purification procedure for a new kind of extradiol dioxygenase, termed chlorocatechol 2,3-dioxygenase, that converts 3-chlorocatechol productively was developed. Structural and kinetic properties of the enzyme, which is part of the degradative pathway used for growth of Pseudomonas putida GJ31 with chlorobenzene, were investigated. The enzyme has a subunit molecular mass of 33.4 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the native Mr value under nondenaturating conditions by gel filtration gave a molecular mass of 135 ± 10 kDa, indicating a homotetrameric enzyme structure (4 × 33.4 kDa). The pI of the enzyme was estimated to be 7.1 ± 0.1. The N-terminal amino acid sequence (43 residues) of the enzyme was determined and exhibits 70 to 42% identity with other extradiol dioxygenases. Fe(II) seems to be a cofactor of the enzyme, as it is for other catechol 2,3-dioxygenases. In contrast to other extradiol dioxygenases, the enzyme exhibited great sensitivity to temperatures above 40°C. The reactivity of this enzyme toward various substituted catechols, especially 3-chlorocatechol, was different from that observed for other catechol 2,3-dioxygenases. Stoichiometric displacement of chloride occurred from 3-chlorocatechol, leading to the production of 2-hydroxymuconate. PMID:9440519

  9. A functional model of extradiol-cleaving catechol dioxygenases: mimicking the 2-his-1-carboxylate facial triad.

    PubMed

    Paria, Sayantan; Halder, Partha; Paine, Tapan Kanti

    2010-05-17

    The synthesis and characterization of an iron-catecholate model complex of a tridentate 2-N-1-carboxylate ligand derived from L-proline are reported. The X-ray crystal structure of the complex [(L)(3)Fe(3)(DBC)(3)] (1) (where L is 1-(2-pyridylmethyl)pyrrolidine-2-carboxylate and DBC is the dianion of 3,5-di-tert-butyl catechol) reveals that the tridentate ligand binds to the iron center in a facial manner and mimics the 2-his-1-carboxylate facial triad motif observed in extradiol-cleaving catechol dioxygenases. The iron(III)-catecholate complex (1) reacts with dioxygen in acetonitrile in ambient conditions to cleave the C-C bond of catecholate. In the reaction, an equal amount of extra- and intradiol cleavage products are formed without any auto-oxidation product. The iron-catecholate complex is a potential functional model of extradiol-cleaving catechol dioxygenases.

  10. Characterization of recombinant Beta vulgaris 4,5-DOPA-extradiol-dioxygenase active in the biosynthesis of betalains.

    PubMed

    Gandía-Herrero, Fernando; García-Carmona, Francisco

    2012-07-01

    Betalains are water-soluble pigments with high antiradical capacity which bestow bright colors to flowers, fruits and other parts of most plants of the order Caryophyllales. The formation of the structural unit of all betalains, betalamic acid from the precursor amino acid 4,5-dihydroxyphenylalanine is catalyzed by the enzyme 4,5-DOPA-extradiol-dioxygenase followed by intramolecular cyclization of the 4,5-secodopa intermediate. This paper describes the purification and the molecular and functional characterization of an active 4,5-DOPA-extradiol-dioxygenase from the best-known source of betalains-Beta vulgaris-after heterologous expression in Escherichia coli. The enzyme is a monomeric protein with a molecular mass of 32 kDa characterized by chromatography, electrophoresis and MALDI-TOF analysis. Enzyme kinetic properties are characterized in the production of betalamic acid, the structural, chromophoric and bioactive unit of plant pigment betalains.

  11. The Complete Reaction Mechanism of Indoleamine 2,3-Dioxygenase as Revealed by QM/MM Simulations

    PubMed Central

    Capece, Luciana; Lewis-Ballester, Ariel; Yeh, Syun-Ru; Estrin, Dario A.; Marti, Marcelo A.

    2012-01-01

    Indoleamine 2,3 dioxygenase (IDO) and tryptophan dioxygenase (TDO) are two heme-proteins that catalyze the oxidation reaction of tryptophan (Trp) to N-formylkynurenine (NFK). Human IDO (hIDO) has recently been recognized as a potent anti-cancer drug target, a fact that triggered intense research on the reaction and inhibition mechanisms of hIDO. Our recent studies revealed that the dioxygenase reaction catalyzed by hIDO and TDO is initiated by addition of the ferric iron-bound superoxide to the C2=C3 bond of Trp to form a ferryl and Trp-epoxide intermediate, via a 2-indolenylperoxo radical transition state. The data demonstrate that the two atoms of dioxygen are inserted into the substrate in a stepwise fashion, challenging the paradigm of heme-based dioxygenase chemistry. In the current study, we used QM/MM methods to decipher the mechanism by which the second ferryl oxygen is inserted into the Trp-epoxide to form the NFK product in hIDO. Our results show that the most energetically favored pathway involves proton transfer from Trp-NH3+ to the epoxide oxygen, triggering epoxide ring-opening and a concerted nucleophilic attack of the ferryl oxygen to the C2 of Trp that leads to a meta-stable reaction intermediate. This intermediate subsequently converts to NFK, following C2-C3 bond cleavage and the associated back proton transfer from the oxygen to the amino group of Trp. A comparative study with Xantomonas campestris TDO (xcTDO) indicates that the reaction follows a similar pathway, although subtle differences distinguishing the two enzyme reactions are evident. The results underscore the importance of the NH3+ group of Trp in the two-step ferryl-based mechanism of hIDO and xcTDO, by acting as an acid catalyst to facilitate the epoxide ring-opening reaction and ferryl oxygen addition to the indole ring. PMID:22196056

  12. Molecular characterization of an inducible gentisate 1,2-dioxygenase gene, xlnE, from Pseudomonas alcaligenes NCIMB 9867.

    PubMed

    Yeo, Chew Chieng; Wong, Mark Vee-Meng; Feng, Yongmei; Song, Keang Peng; Poh, Chit Laa

    2003-07-17

    Pseudomonas alcaligenes NCIMB 9867 (strain P25X) produces isofunctional enzymes of the gentisate pathway that enables the degradation of xylenols and cresols via gentisate. Previous reports had indicated that one set of enzymes is constitutively expressed whereas the other set is strictly inducible by aromatic hydrocarbon substrates. The gene encoding gentisate 1,2-dioxygenase (GDO), the enzyme that catalyzes the cleavage of the gentisate aromatic ring, was cloned from strain P25X. The GDO gene, designated xlnE, is 1,044 bp, and is part of a 5.4 kb operon which consists of six genes, xlnEFGHID. Transcription of this operon was driven by a sigma 70-type promoter, PxlnE, located 123 bp upstream of the xlnE start codon. Primer extension analysis showed that the xlnE transcription start point is located at the -87 adenine residue. In a P25X xlnE knockout mutant, GDO activity could only be detected when cells were grown in the presence of aromatic substrates, suggesting that xlnE encodes for the constitutive copy of GDO. This was verified by constructing a P25X strain with xlnE transcriptionally fused to a promoterless catechol 2,3-dioxygenase gene. In this strain, catechol 2,3-dioxygenase activity was detected in cells that were grown in the absence of aromatic inducers. However, catechol 2,3-dioxygenase activity increased up to four fold when these cells were grown in the presence of aromatic substrates, in particular 3-hydroxybenzoate. Thus, xlnE is in fact, inducible and the constitutive activity observed under non-inducing conditions was due to its relatively high basal levels of expression.

  13. The gene coding for the DOPA dioxygenase involved in betalain biosynthesis in Amanita muscaria and its regulation.

    PubMed

    Hinz, U G; Fivaz, J; Girod, P A; Zyrd, J P

    1997-09-01

    Genomic and cDNA clones derived from the gene (dodA) coding for DOPA dioxygenase, a key enzyme in the betalain pathway, were obtained from the basidiomycete Amanita muscaria. A cDNA library was established in the phage lambda ZapII and dodA clones were isolated using polyclonal antibodies raised against the purified enzyme. Their identity was confirmed by comparison of the deduced amino acid sequence with the sequence of several tryptic peptide fragments of DOPA dioxygenase. The gene coded for a 228-amino acid protein that showed no homology to published sequences. The coding region was interrupted by five short introns. Regulation was shown to occur at the transcriptional level; the mRNA accumulated to high levels only in the coloured cap tissue. dodA was found to be a single-copy gene in A. muscaria. To our knowledge, this is the first gene from the betalain pathway to be cloned. It encodes a type of aromatic ring-cleaving dioxygenase that has not been previously described.

  14. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    DOE PAGES

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog ofmore » uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.« less

  15. Structures of Arg- and Gln-type bacterial cysteine dioxygenase homologs: Arg- and Gln-type Bacterial CDO Homologs

    SciTech Connect

    Driggers, Camden M.; Hartman, Steven J.; Karplus, P. Andrew

    2015-01-01

    In some bacteria, cysteine is converted to cysteine sulfinic acid by cysteine dioxygenases (CDO) that are only ~15–30% identical in sequence to mammalian CDOs. Among bacterial proteins having this range of sequence similarity to mammalian CDO are some that conserve an active site Arg residue (“Arg-type” enzymes) and some having a Gln substituted for this Arg (“Gln-type” enzymes). Here, we describe a structure from each of these enzyme types by analyzing structures originally solved by structural genomics groups but not published: a Bacillus subtilis “Arg-type” enzyme that has cysteine dioxygenase activity (BsCDO), and a Ralstonia eutropha “Gln-type” CDO homolog of uncharacterized activity (ReCDOhom). The BsCDO active site is well conserved with mammalian CDO, and a cysteine complex captured in the active site confirms that the cysteine binding mode is also similar. The ReCDOhom structure reveals a new active site Arg residue that is hydrogen bonding to an iron-bound diatomic molecule we have interpreted as dioxygen. Notably, the Arg position is not compatible with the mode of Cys binding seen in both rat CDO and BsCDO. As sequence alignments show that this newly discovered active site Arg is well conserved among “Gln-type” CDO enzymes, we conclude that the “Gln-type” CDO homologs are not authentic CDOs but will have substrate specificity more similar to 3-mercaptopropionate dioxygenases.

  16. An aryl dioxygenase shows remarkable double dioxygenation capacity for diverse bis-aryl compounds, provided they are carbocyclic.

    PubMed

    Overwin, Heike; González, Myriam; Méndez, Valentina; Seeger, Michael; Wray, Victor; Hofer, Bernd

    2016-09-01

    The bacterial dioxygenation of mono- or polycyclic aromatic compounds is an intensely studied field. However, only in a few cases has the repeated dioxygenation of a substrate possessing more than a single aromatic ring been described. We previously characterized the aryl-hydroxylating dioxygenase BphA-B4h, an artificial hybrid of the dioxygenases of the biphenyl degraders Burkholderia xenovorans LB400 and Pseudomonas sp. strain B4-Magdeburg, which contains the active site of the latter enzyme, as an exceptionally powerful biocatalyst. We now show that this dioxygenase possesses a remarkable capacity for the double dioxygenation of various bicyclic aromatic compounds, provided that they are carbocyclic. Two groups of biphenyl analogues were examined: series A compounds containing one heterocyclic aromatic ring and series B compounds containing two homocyclic aromatic rings. Whereas all of the seven partially heterocyclic biphenyl analogues were solely dioxygenated in the homocyclic ring, four of the six carbocyclic bis-aryls were converted into ortho,meta-hydroxylated bis-dihydrodiols. Potential reasons for failure of heterocyclic dioxygenations are discussed. The obtained bis-dihydrodiols may, as we also show here, be enzymatically re-aromatized to yield the corresponding tetraphenols. This opens a way to a range of new polyphenolic products, a class of compounds known to exert multiple biological activities. Several of the obtained compounds are novel molecules.

  17. Crystallization and preliminary crystallographic analysis of the catechol 2,3-dioxygenase PheB from Bacillus stearothermophilus BR219

    SciTech Connect

    Sugimoto, Keisuke; Matsufuzi, Kazuki; Ohnuma, Hiroaki; Senda, Miki; Fukuda, Masao; Senda, Toshiya

    2006-02-01

    PheB, an extradiol-cleaving catecholic dioxygenase, was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystal belongs to the orthorhombic system, space group P2{sub 1}2{sub 1}2{sub 1}, and diffracts to 2.3 Å resolution. Class II extradiol-cleaving catecholic dioxygenase, a key enzyme of aromatic compound degradation in bacteria, cleaves the aromatic ring of catechol by adding two O atoms. PheB is one of the class II extradiol-cleaving catecholic dioxygenases and shows a high substrate specificity for catechol derivatives, which have one aromatic ring. In order to reveal the mechanism of the substrate specificity of PheB, PheB has been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The space group of the obtained crystal was P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 65.5, b = 119.2, c = 158.7 Å. The crystal diffracted to 2.3 Å resolution.

  18. Structural Basis of the Enhanced Pollutant-Degrading Capabilities of an Engineered Biphenyl Dioxygenase

    PubMed Central

    Dhindwal, Sonali; Gomez-Gil, Leticia; Neau, David B.; Pham, Thi Thanh My; Sylvestre, Michel; Eltis, Lindsay D.; Bolin, Jeffrey T.

    2016-01-01

    ABSTRACT Biphenyl dioxygenase, the first enzyme of the biphenyl catabolic pathway, is a major determinant of which polychlorinated biphenyl (PCB) congeners are metabolized by a given bacterial strain. Ongoing efforts aim to engineer BphAE, the oxygenase component of the enzyme, to efficiently transform a wider range of congeners. BphAEII9, a variant of BphAELB400 in which a seven-residue segment, 335TFNNIRI341, has been replaced by the corresponding segment of BphAEB356, 333GINTIRT339, transforms a broader range of PCB congeners than does either BphAELB400 or BphAEB356, including 2,6-dichlorobiphenyl, 3,3′-dichlorobiphenyl, 4,4′-dichlorobiphenyl, and 2,3,4′-trichlorobiphenyl. To understand the structural basis of the enhanced activity of BphAEII9, we have determined the three-dimensional structure of this variant in substrate-free and biphenyl-bound forms. Structural comparison with BphAELB400 reveals a flexible active-site mouth and a relaxed substrate binding pocket in BphAEII9 that allow it to bind different congeners and which could be responsible for the enzyme's altered specificity. Biochemical experiments revealed that BphAEII9 transformed 2,3,4′-trichlorobiphenyl and 2,2′,5,5′-tetrachlorobiphenyl more efficiently than did BphAELB400 and BphAEB356. BphAEII9 also transformed the insecticide dichlorodiphenyltrichloroethane (DDT) more efficiently than did either parental enzyme (apparent kcat/Km of 2.2 ± 0.5 mM−1 s−1, versus 0.9 ± 0.5 mM−1 s−1 for BphAEB356). Studies of docking of the enzymes with these three substrates provide insight into the structural basis of the different substrate selectivities and regiospecificities of the enzymes. IMPORTANCE Biphenyl dioxygenase is the first enzyme of the biphenyl degradation pathway that is involved in the degradation of polychlorinated biphenyls. Attempts have been made to identify the residues that influence the enzyme activity for the range of substrates among various species. In this study

  19. Characterization of three distinct extradiol dioxygenases involved in mineralization of dibenzofuran by Terrabacter sp. strain DPO360.

    PubMed

    Schmid, A; Rothe, B; Altenbuchner, J; Ludwig, W; Engesser, K H

    1997-01-01

    The dibenzofuran-degrading bacterial strain DPO360 represents a new species of the genus Terrabacter together with the previously described dibenzofuran-mineralizing bacterial strain DPO1361 (K.-H. Engesser, V. Strubel, K. Christoglou, P. Fischer, and H. G. Rast, FEMS Microbiol. Lett. 65:205-210, 1989; V. Strubel, Ph.D. thesis, University of Stuttgart, Stuttgart, Germany, 1991; V. Strubel, H. G. Rast, W. Fietz, H.-J. Knackmuss, and K.-H. Engesser, FEMS Microbiol. Lett. 58:233-238, 1989). Two 2,3-dihydroxybiphenyl-1,2-dioxygenases (BphC1 and BphC2) and one catechol-2,3-dioxygenase (C23O) were shown to be expressed in Terrabacter sp. strain DPO360 growing with dibenzofuran as a sole source of carbon and energy. These enzymes exhibited strong sensitivity to oxygen. They were purified to apparent homogeneity as homodimers (BphC and BphC2) and as a homotetrameric catechol-2,3-dioxygenase (C23O). According to their specificity constants kcat/Km, both BphC1 and BphC2 were shown to be responsible for the cleavage of 2,2',3-trihydroxybiphenyl, the first metabolite in dibenzofuran mineralization along the angular dioxygenation pathway. With this substrate, BphC2 exhibited a considerably higher kcat/Km, value (183 microM/min) than BphC1 (29 microM/min). Catechol-2,3-dioxygenase was recognized to be not involved in the ring cleavage of 2,2',3-trihydroxybiphenyl (kcat/Km, 1 microM/min). Analysis of deduced amino acid sequence data of bphC1 revealed 36% sequence identity to nahC from Pseudomonas putida PpG7 (S. Harayama and M. Rekik, J. Biol. Chem. 264:15328-15333, 1989) and about 40% sequence identity to various bphC genes from different Pseudomonas and Rhodococcus strains. In addition, another 2,3-dihydroxybiphenyl-1,2-dioxygenase gene (bphC3) was cloned from the genome of Terrabacter sp. strain DPO360. Expression of this gene, however, could not be detected in Terrabacter sp. strain DPO360 after growth with dibenzofuran.

  20. Degradation of diphenyl ether in Sphingobium phenoxybenzoativorans SC_3 is initiated by a novel ring-cleavage dioxygenase.

    PubMed

    Cai, Shu; Chen, Li-Wei; Ai, Yu-Chun; Qiu, Ji-Guo; Wang, Cheng-Hong; Shi, Chao; He, Jian; Cai, Tian-Ming

    2017-03-10

    Sphingobium phenoxybenzoativorans SC_3 degrades and utilizes diphenyl ether (DE) and 2-carboxy DE as its sole carbon and energy source. In this study, we report the degradation of DE and 2-carboxy DE initiated by a novel ring-cleavage angular dioxygenase (Dpe) in the strain. Dpe functions at the angular carbon and its adjacent carbon (C1a, C2) of a benzene ring in DE (or the 2-carboxy benzene ring in 2-carboxy DE) and cleaves the C1a-C2 bond (decarboxylation is simultaneously happened for 2-carboxy DE), yielding 2,4-hexadienal phenyl ester, which is subsequently hydrolyzed to muconic acid semialdehyde and phenol. Dpe is a type IV Rieske non-heme iron oxygenase (RHO) and consists of three components: a hetero-oligomer oxygenase, a [2Fe-2S]-type ferredoxin and a GR (glutathione reductase)-type reductase. Genetic analyses revealed that dpeA1A2 plays an essential role in degradation and utilization of DE and 2-carboxy DE in S. phenoxybenzoativorans SC_3. Enzymatic study showed that transformation of one molecule of DE needs two molecules of oxygen and two molecules of NADH, supporting the assumption that the cleavage of DE catalyzed by Dpe is a continuous two-step dioxygenation process: DE is dioxygenated at C1a, C2 to form an hemiacetal-like intermediates, which is further dioxygenated resulting the cleavage of the C1a-C2 bond to form one molecule of 2,4-hexadienal phenyl ester and two molecules of H2O. This study extends our knowledge of the mode and mechanism of ring-cleavage of aromatic compounds.IMPORTANCE Benzene ring-cleavage, catalyzed by dioxygenase, is the key and speed limiting step in the aerobic degradation of aromatic compounds. Previously reported ring-cleavage of DEs, the benzene ring needs to be firstly dihydroxylated at lateral position, and subsequently dehydrogenated and opened through extradiol cleavage. This process requires three enzymes (two dioxygenases and one dehydrogenase). In this study, we identified a novel angular dioxygenase (Dpe) in S

  1. Regulation of indoleamine 2,3-dioxygenase in primary human saphenous vein endothelial cells

    PubMed Central

    Mouratidis, Petros XE; George, Andrew JT

    2015-01-01

    Background Indoleamine 2,3-dioxygenase (IDO) is an enzyme associated with the regulation of immune responses. Cytokines such as IFNγ induce its expression in endothelial cells originating from immune-privileged sites. In this study, we investigate regulators of IDO in primary endothelial cells from a non-immune-privileged site and determine whether IDO expression affects immune cell behavior. Methods IDO expression was determined using real-time quantitative polymerase chain reaction and immunoblotting. IDO activity was estimated using an IDO enzyme assay. Primary cells were transfected using microporation, and T-cell migration was determined using a cell transmigration assay. Results IDO is expressed in human saphenous vein endothelial cells after stimulation with IFNγ but not after treatment with TNFα, IL-1β, IL-2, IL-4, IL-6, or IL-10. VEGFβ and heparin negatively regulate IFNγ-driven increases in IDO. Overexpression of IDO in endothelial cells does not affect transmigration of T-cells. Conclusion IDO is expressed in human saphenous vein endothelial cells after stimulation with IFNγ. Heparin and angiogenesis stimulators such as VEGFβ negatively regulate its expression. PMID:26056484

  2. Emerging concepts on inhibitors of indoleamine 2,3-dioxygenase in rheumatic diseases.

    PubMed

    Filippini, P; Del Papa, N; Sambataro, D; Del Bufalo, A; Locatelli, F; Rutella, S

    2012-01-01

    The enzyme indoleamine 2,3-dioxygenase 1 (IDO1) finely regulates both innate and adaptive immune responses through the degradation of the essential amino acid tryptophan into kynurenine and other downstream metabolites, which suppress effector T-cell function and promote the differentiation of regulatory T cells. A novel role for IDO1 as a signaling molecule and a modifier of innate inflammatory responses is now emerging. In particular, IDO1 can either support or antagonize inflammation in a context- and tissuedependent manner. Studies in experimental arthritis have unravelled a previously unappreciated role for IDO in controlling B-cell activation and autoantibody production. IDO dysregulation has been documented in patients with systemic lupus erythematosus, systemic sclerosis and Sjogren's syndrome, as well as in severe sepsis and chronic kidney disease. This article summarizes the contribution of IDO to the pathophysiology of inflammatory/autoimmune disorders, and discusses whether strategies to restore metabolic equilibrium in the kynurenine pathway might be pursued in diseases states such as rheumatoid arthritis and systemic sclerosis.

  3. Cysteine dioxygenase type 1 promotes adipogenesis via interaction with peroxisome proliferator-activated receptor gamma

    SciTech Connect

    Deng, Peng; Chen, Yi; Ji, Ning; Lin, Yunfeng; Yuan, Quan; Ye, Ling; Chen, Qianming

    2015-02-27

    Mammalian cysteine dioxygenase type 1 (CDO1) is an essential enzyme for taurine biosynthesis and the biodegradation of toxic cysteine. As previously suggested, Cdo1 may be a marker of liposarcoma progression and adipogenic differentiation, but the role of Cdo1 in adipogenesis has yet been reported. In this study, we found that the expression of Cdo1 is dramatically elevated during adipogenic differentiation of 3T3-L1 pre-adipocytes and mouse bone marrow-derived mesenchymal stem cells (mBMSCs). Conversely, knockdown of Cdo1 inhibited expression of adipogenic specific genes and lipid droplet formation in 3T3-L1 cells and mBMSCs. Mechanistically, we found Cdo1 interacted with Pparγ in response to adipogenic stimulus. Further, depletion of Cdo1 reduced the recruitment of Pparγ to the promoters of C/EBPα and Fabp4. Collectively, our finding indicates that Cdo1 may be a co-activator of Pparγ in adipogenesis, and may contribute to the development of disease associated with excessive adipose tissue. - Highlights: • Cdo1expression is highly up-regulated during adipogenic differentiation of 3T3-L1 and mBMSCs. • Depletion of Cdo1 inhibited expression of adipogenic specific genes and lipid droplet formation. • Cdo1interacts with Pparγ during adipogenesis. • Knockdown of Cdo1 inhibited Pparγ binding to the promoters of C/EBPα and Fabp4.

  4. Extradiol dioxygenase-SiO₂ sol-gel modified electrode for catechol and its derivatives detection.

    PubMed

    Zhang, Qiang; Qu, Yuanyuan; Zhang, Xuwang; Zhou, Jiti; Wang, Hongtao

    2011-07-15

    A feasible and sensitive biosensor for catechol and its derivatives using 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC)-modified glassy carbon electrode was successfully constructed by polyvinyl alcohol-modified SiO₂ sol-gel method. The as-prepared biosensor was characterized by electrochemical impedance spectroscopy, and the surface topography of the film was imaged by atomic force microscope. Liquid chromatography-tandem mass spectrometry was applied to reveal the catalytic mechanism. BphC embedded in SiO₂ gel maintained its bioactivity well and exhibited excellent eletrocatalytical response to both catechol and some of its derivatives (such as 3-methylcatechol and 4-methylcatechol). The biosensor showed a linear amperometric response range between 0.002 mM and 0.8 mM catechol. And the sensitivity was 1.268 mA/(mM cm²) with a detection limit of 0.428 μM for catechol (S/N = 3). Furthermore, the BphC biosensor exhibited perfect selectivity for catechol in the mixtures of catechol and phenol. It was suggested that this flexible protocol would open up a new avenue for designing other ring-cleavage enzyme biosensors, which could be widely used for monitoring various kinds of environmental pollutants.

  5. Indoleamine 2,3 Dioxygenase as a Potential Therapeutic Target in Huntington’s Disease

    PubMed Central

    Mazarei, Gelareh; Leavitt, Blair R.

    2015-01-01

    Abstract Within the past decade, there has been increasing interest in the role of tryptophan (Trp) metabolites and the kynurenine pathway (KP) in diseases of the brain such as Huntington’s disease (HD). Evidence is accumulating to suggest that this pathway is imbalanced in neurologic disease states. The KP diverges into two branches that can lead to production of either neuroprotective or neurotoxic metabolites. In one branch, kynurenine (Kyn) produced as a result of tryptophan (Trp) catabolism is further metabolized to neurotoxic metabolites such as 3-hydroxykunurenine (3-HK) and quinolinic acid (QA). In the other branch, Kyn is converted to the neuroprotective metabolite kynurenic acid (KA). The enzyme Indoleamine 2,3 dioxygenase (IDO1) catalyzes the conversion of Trp into Kyn, the first and rate-limiting enzymatic step of the KP. This reaction takes place throughout the body in multiple cell types as a required step in the degradation of the essential amino acid Trp. Studies of IDO1 in brain have focused primarily on a potential role in depression, immune tolerance associated with brain tumours, and multiple sclerosis; however the role of this enzyme in neurodegenerative disease has garnered significant attention in recent years. This review will provide a summary of the current understanding of the role of IDO1 in Huntington’s disease and will assess this enzyme as a potential therapeutic target for HD. PMID:26397892

  6. Indoleamine 2,3 Dioxygenase as a Potential Therapeutic Target in Huntington's Disease.

    PubMed

    Mazarei, Gelareh; Leavitt, Blair R

    2015-01-01

    Within the past decade, there has been increasing interest in the role of tryptophan (Trp) metabolites and the kynurenine pathway (KP) in diseases of the brain such as Huntington's disease (HD). Evidence is accumulating to suggest that this pathway is imbalanced in neurologic disease states. The KP diverges into two branches that can lead to production of either neuroprotective or neurotoxic metabolites. In one branch, kynurenine (Kyn) produced as a result of tryptophan (Trp) catabolism is further metabolized to neurotoxic metabolites such as 3-hydroxykunurenine (3-HK) and quinolinic acid (QA). In the other branch, Kyn is converted to the neuroprotective metabolite kynurenic acid (KA). The enzyme Indoleamine 2,3 dioxygenase (IDO1) catalyzes the conversion of Trp into Kyn, the first and rate-limiting enzymatic step of the KP. This reaction takes place throughout the body in multiple cell types as a required step in the degradation of the essential amino acid Trp. Studies of IDO1 in brain have focused primarily on a potential role in depression, immune tolerance associated with brain tumours, and multiple sclerosis; however the role of this enzyme in neurodegenerative disease has garnered significant attention in recent years. This review will provide a summary of the current understanding of the role of IDO1 in Huntington's disease and will assess this enzyme as a potential therapeutic target for HD.

  7. The Role of Indoleamine 2, 3-Dioxygenase in Immune Suppression and Autoimmunity

    PubMed Central

    Mbongue, Jacques C.; Nicholas, Dequina A.; Torrez, Timothy W.; Kim, Nan-Sun; Firek, Anthony F.; Langridge, William H.R.

    2015-01-01

    Indoleamine 2, 3-dioxygenase (IDO) is the first and rate limiting catabolic enzyme in the degradation pathway of the essential amino acid tryptophan. By cleaving the aromatic indole ring of tryptophan, IDO initiates the production of a variety of tryptophan degradation products called “kynurenines” that are known to exert important immuno-regulatory functions. Because tryptophan must be supplied in the diet, regulation of tryptophan catabolism may exert profound effects by activating or inhibiting metabolism and immune responses. Important for survival, the regulation of IDO biosynthesis and its activity in cells of the immune system can critically alter their responses to immunological insults, such as infection, autoimmunity and cancer. In this review, we assess how IDO-mediated catabolism of tryptophan can modulate the immune system to arrest inflammation, suppress immunity to cancer and inhibit allergy, autoimmunity and the rejection of transplanted tissues. Finally, we examine how vaccines may enhance immune suppression of autoimmunity through the upregulation of IDO biosynthesis in human dendritic cells. PMID:26378585

  8. Niacin metabolism and indoleamine 2,3-dioxygenase activation in malnourished patients with flaky paint dermatosis.

    PubMed

    Maltos, André Luiz; Portari, Guilherme Vannucchi; Moraes, Giselle Vanessa; Monteiro, Marina Casteli Rodrigues; Vannucchi, Helio; da Cunha, Daniel Ferreira

    2015-06-01

    Flaky paint dermatosis, characterized by extensive, often bilateral areas of flaking and pigmentation, mostly in sun unexposed areas is considered a feature of kwashiorkor in both children and adults, and must be differentiated from other dermatosis, including chapped and xerotica skin, and pellagra. In this case series we provide evidence that malnourished patients with flaky paint dermatosis and infection/inflammation shown laboratory data suggestive of indoleamine 2,3-dioxygenase (IDO) activation, besides decreased urinary excretion of N1-methylnicotinamide (N1 MN), a marker of pellagra. We study nine adult patients showing flaky paint dermatosis and clinical features of infection or inflammation, and increased serum C-reactive protein, characteristic of the presence of acute phase response syndrome. As a group, they had low or deficient urinary N1 MN excretion (0.52 ± 0.39 mg/g creatinine) compatible with pellagra. They also showed low serum tryptophan levels (<29 μmol/L) and a serum kynurenine/tryptophan ratio higher than 0.04, suggesting increased IDO expression and increase in the tryptophan oxidation. Findings suggest that some patients with flaky paint dermatosis showed laboratory data suggestive of IDO activation, besides decreased N1 MN urinary excretion. Taken together, the data support the idea that flaky paint dermatosis could be a skin manifestation of niacin deficiency.

  9. Differential spatio-temporal expression of carotenoid cleavage dioxygenases regulates apocarotenoid fluxes during AM symbiosis.

    PubMed

    López-Ráez, Juan A; Fernández, Iván; García, Juan M; Berrio, Estefanía; Bonfante, Paola; Walter, Michael H; Pozo, María J

    2015-01-01

    Apocarotenoids are a class of compounds that play important roles in nature. In recent years, a prominent role for these compounds in arbuscular mycorrhizal (AM) symbiosis has been shown. They are derived from carotenoids by the action of the carotenoid cleavage dioxygenase (CCD) enzyme family. In the present study, using tomato as a model, the spatio-temporal expression pattern of the CCD genes during AM symbiosis establishment and functioning was investigated. In addition, the levels of the apocarotenoids strigolactones (SLs), C13 α-ionol and C14 mycorradicin (C13/C14) derivatives were analyzed. The results suggest an increase in SLs promoted by the presence of the AM fungus at the early stages of the interaction, which correlated with an induction of the SL biosynthesis gene SlCCD7. At later stages, induction of SlCCD7 and SlCCD1 expression in arbusculated cells promoted the production of C13/C14 apocarotenoid derivatives. We show here that the biosynthesis of apocarotenoids during AM symbiosis is finely regulated throughout the entire process at the gene expression level, and that CCD7 constitutes a key player in this regulation. Once the symbiosis is established, apocarotenoid flux would be turned towards the production of C13/C14 derivatives, thus reducing SL biosynthesis and maintaining a functional symbiosis.

  10. Nanomedicine and cancer immunotherapy: focus on indoleamine 2,3-dioxygenase inhibitors

    PubMed Central

    Zulfiqar, Bilal; Mahroo, Amnah; Nasir, Kaenat; Farooq, Rai Khalid; Jalal, Nasir; Rashid, Muhammad Usman; Asghar, Kashif

    2017-01-01

    Nanomedicine application in cancer immunotherapy is currently one of the most challenging areas in cancer therapeutic intervention. Innovative solutions have been provided by nanotechnology to deliver cytotoxic agents to the cancer cells partially affecting the healthy cells of the body during the process. Nanoparticle-based drug delivery is an emerging approach to stimulate the immune responses against cancer. The inhibition of indoleamine 2,3-dioxygenase (IDO) is a pivotal area of research in cancer immunotherapy. IDO is a heme-containing immunosuppressive enzyme, which is responsible for the degradation of tryptophan while increasing the concentration of kynurenine metabolites. Various preclinical studies showed that IDO inhibition in certain diseases may result in significant therapeutic effects. Here, we provide a review of the natural and synthetic inhibitors of IDO. These inhibitors are classified according to their source, inhibitory concentrations, the chemical structure, and the mechanism of action. Tumor-targeted chemotherapy is an advanced technique and has more advantages as compared to the conventional chemotherapy. Search for more efficient and less toxic nanoparticles in conjunction with compounds to inhibit IDO is still an area of interest for several research groups worldwide, especially revealing to be an extensive and a promising area in cancer therapeutic innovations. PMID:28176942

  11. Catalytic Activity of Human Indoleamine 2,3-Dioxygenase (hIDO1) at Low Oxygen

    PubMed Central

    Kolawole, Ayodele O.; Hixon, Brian P.; Dameron, Laura S.; Chrisman, Ian M.; Smirnov, Valeriy V.

    2015-01-01

    A cytokine-inducible extrahepatic human indoleamine 2,3-dioxygenase (hIDO1) catalyzes the first step of the kynurenine pathway. Immunosuppressive activity of hIDO1 in tumor cells weakens host T-cell immunity, contributing to the progression of cancer. Here we report on enzyme kinetics and catalytic mechanism of hIDO1, studied at varied levels of dioxygen (O2) and L-tryptophan (L-Trp). Using a cytochrome b5-based activating system, we measured the initial rates of O2 decay with a Clark-type oxygen electrode at physiologically-relevant levels of both substrates. Kinetics was also studied in the presence of two substrate analogs: 1-methyl-L-tryptophan and norharmane. Quantitative analysis supports a steady-state rather than a rapid equilibrium kinetic mechanism, where the rates of individual pathways, leading to a ternary complex, are significantly different, and the overall rate of catalysis depends on contributions of both routes. One path, where O2 binds to ferrous hIDO1 first, is faster than the second route, which starts with the binding of L-Trp. However, L-Trp complexation with free ferrous hIDO1 is more rapid than that of O2. As the level of L-Trp increases, the slower route becomes a significant contributor to the overall rate, resulting in observed substrate inhibition. PMID:25712221

  12. Characterization of the Role of β-Carotene 9,10-Dioxygenase in Macular Pigment Metabolism*

    PubMed Central

    Babino, Darwin; Palczewski, Grzegorz; Widjaja-Adhi, M. Airanthi K.; Kiser, Philip D.; Golczak, Marcin; von Lintig, Johannes

    2015-01-01

    A family of enzymes collectively referred to as carotenoid cleavage oxygenases is responsible for oxidative conversion of carotenoids into apocarotenoids, including retinoids (vitamin A and its derivatives). A member of this family, the β-carotene 9,10-dioxygenase (BCO2), converts xanthophylls to rosafluene and ionones. Animals deficient in BCO2 highlight the critical role of the enzyme in carotenoid clearance as accumulation of these compounds occur in tissues. Inactivation of the enzyme by a four-amino acid-long insertion has recently been proposed to underlie xanthophyll concentration in the macula of the primate retina. Here, we focused on comparing the properties of primate and murine BCO2s. We demonstrate that the enzymes display a conserved structural fold and subcellular localization. Low temperature expression and detergent choice significantly affected binding and turnover rates of the recombinant enzymes with various xanthophyll substrates, including the unique macula pigment meso-zeaxanthin. Mice with genetically disrupted carotenoid cleavage oxygenases displayed adipose tissue rather than eye-specific accumulation of supplemented carotenoids. Studies in a human hepatic cell line revealed that BCO2 is expressed as an oxidative stress-induced gene. Our studies provide evidence that the enzymatic function of BCO2 is conserved in primates and link regulation of BCO2 gene expression with oxidative stress that can be caused by excessive carotenoid supplementation. PMID:26307071

  13. Negative Impact of Hypoxia on Tryptophan 2,3-Dioxygenase Function

    PubMed Central

    Elbers, Frank; Woite, Claudia; Antoni, Valentina; Stein, Sara; Funakoshi, Hiroshi; Nakamura, Toshikazu; Schares, Gereon; Däubener, Walter

    2016-01-01

    Tryptophan is an essential amino acid for hosts and pathogens. The liver enzyme tryptophan 2,3-dioxygenase (TDO) provokes, by its ability to degrade tryptophan to N-formylkynurenine, the precursor of the immune-relevant kynurenines, direct and indirect antimicrobial and immunoregulatory states. Up to now these TDO-mediated broad-spectrum effector functions have never been observed under hypoxia in vitro, although physiologic oxygen concentrations in liver tissue are low, especially in case of infection. Here we analysed recombinant expressed human TDO and ex vivo murine TDO functions under different oxygen conditions and show that TDO-induced restrictions of clinically relevant pathogens (bacteria, parasites) and of T cell proliferation are abrogated under hypoxic conditions. We pinpointed the loss of TDO efficiency to the reduction of TDO activity, since cell survival and TDO protein levels were unaffected. In conclusion, the potent antimicrobial as well as immunoregulatory effects of TDO were substantially impaired under hypoxic conditions that pathophysiologically occur in vivo. This might be detrimental for the appropriate host immune response towards relevant pathogens. PMID:27563172

  14. Toluene and ethylbenzene oxidation by purified naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

    PubMed

    Lee, K; Gibson, D T

    1996-09-01

    Purified naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized toluene to benzyl alcohol and benzaldehyde by reactions involving benzylic monooxygenation and dioxygen-dependent alcohol oxidation, respectively. Xylene and nitrotoluene isomers were also oxidized to substituted benzyl alcohol and benzaldehyde derivatives. NDO oxidized ethylbenzene sequentially through (S)-1-phenethyl alcohol (77% enantiomeric excess) and acetophenone to 2-hydroxyacetophenone. In addition, NDO also oxidized ethylbenzene through styrene to (R)-1-phenyl-1,2-ethanediol (74% enantiomeric excess) by reactions involving desaturation and dihydroxylation, respectively. Isotope experiments with 18O2, H2 18O, and D2O suggest that 1-phenethyl alcohol is oxidized to acetophenone by a minor reaction involving desaturation followed by tautomerization. The major reaction in the conversion of 1-phenethyl alcohol and benzyl alcohol to acetophenone and benzaldehyde, respectively, probably involves monohydroxylation to form a gem-diol intermediate which stereospecifically loses the incoming hydroxyl group to leave the carbonyl product. These results are compared with similar reactions catalyzed by cytochrome P-450.

  15. Tolerance to apoptotic cells is regulated by indoleamine 2,3-dioxygenase

    PubMed Central

    Ravishankar, Buvana; Liu, Haiyun; Shinde, Rahul; Chandler, Phillip; Baban, Babak; Tanaka, Masato; Munn, David H.; Mellor, Andrew L.; Karlsson, Mikael C. I.; McGaha, Tracy L.

    2012-01-01

    Tolerance to self-antigens present in apoptotic cells is critical to maintain immune-homeostasis and prevent systemic autoimmunity. However, mechanisms that sustain self-tolerance are poorly understood. Here we show that systemic administration of apoptotic cells to mice induced splenic expression of the tryptophan catabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO expression was confined to the splenic marginal zone and was abrogated by depletion of CD169+ cells. Pharmacologic inhibition of IDO skewed the immune response to apoptotic cells, resulting in increased proinflammatory cytokine production and increased effector T-cell responses toward apoptotic cell-associated antigens. Presymptomatic lupus-prone MRLlpr/lpr mice exhibited abnormal elevated IDO expression in the marginal zone and red pulp and inhibition of IDO markedly accelerated disease progression. Moreover, chronic exposure of IDO-deficient mice to apoptotic cells induced a lupus-like disease with serum autoreactivity to double-stranded DNA associated with renal pathology and increased mortality. Thus, IDO limits innate and adaptive immunity to apoptotic self-antigens and IDO-mediated regulation inhibits inflammatory pathology caused by systemic autoimmune disease. PMID:22355111

  16. Nitric-oxide dioxygenase function of human cytoglobin with cellular reductants and in rat hepatocytes.

    PubMed

    Gardner, Anne M; Cook, Matthew R; Gardner, Paul R

    2010-07-30

    Cytoglobin (Cygb) was investigated for its capacity to function as a NO dioxygenase (NOD) in vitro and in hepatocytes. Ascorbate and cytochrome b(5) were found to support a high NOD activity. Cygb-NOD activity shows respective K(m) values for ascorbate, cytochrome b(5), NO, and O(2) of 0.25 mm, 0.3 microm, 40 nm, and approximately 20 microm and achieves a k(cat) of 0.5 s(-1). Ascorbate and cytochrome b(5) reduce the oxidized Cygb-NOD intermediate with apparent second order rate constants of 1000 m(-1) s(-1) and 3 x 10(6) m(-1) s(-1), respectively. In rat hepatocytes engineered to express human Cygb, Cygb-NOD activity shows a similar k(cat) of 1.2 s(-1), a K(m)(NO) of 40 nm, and a k(cat)/K(m)(NO) (k'(NOD)) value of 3 x 10(7) m(-1) s(-1), demonstrating the efficiency of catalysis. NO inhibits the activity at [NO]/[O(2)] ratios >1:500 and limits catalytic turnover. The activity is competitively inhibited by CO, is slowly inactivated by cyanide, and is distinct from the microsomal NOD activity. Cygb-NOD provides protection to the NO-sensitive aconitase. The results define the NOD function of Cygb and demonstrate roles for ascorbate and cytochrome b(5) as reductants.

  17. Dioxygenase-mediated quenching of quinolone-dependent quorum sensing in Pseudomonas aeruginosa.

    PubMed

    Pustelny, Christian; Albers, Alexander; Büldt-Karentzopoulos, Klaudia; Parschat, Katja; Chhabra, Siri Ram; Cámara, Miguel; Williams, Paul; Fetzner, Susanne

    2009-12-24

    2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS) is a quorum-sensing signal molecule used by Pseudomonas aeruginosa. The structural similarity between 3-hydroxy-2-methyl-4(1H)-quinolone, the natural substrate for the 2,4-dioxygenase, Hod, and PQS prompted us to investigate whether Hod quenched PQS signaling. Hod is capable of catalyzing the conversion of PQS to N-octanoylanthranilic acid and carbon monoxide. In P. aeruginosa PAO1 cultures, exogenously supplied Hod protein reduced expression of the PQS biosynthetic gene pqsA, expression of the PQS-regulated virulence determinants lectin A, pyocyanin, and rhamnolipids, and virulence in planta. However, the proteolytic cleavage of Hod by extracellular proteases, competitive inhibition by the PQS precursor 2-heptyl-4(1H)-quinolone, and PQS binding to rhamnolipids reduced the efficiency of Hod as a quorum-quenching agent. Nevertheless, these data indicate that enzyme-mediated PQS inactivation has potential as an antivirulence strategy against P. aeruginosa.

  18. Indoleamine 2,3 dioxygenase and regulation of T cell immunity

    SciTech Connect

    Mellor, Andrew . E-mail: amellor@mcg.edu

    2005-12-09

    Regulation of adaptive immune responses is critically important to allow the adaptive immune system to eradicate infections while causing minimal collateral damage to infected tissues, as well as preventing autoimmune disease mediated by self-reactive lymphocytes. Tumors and pathogens that cause persistent infections can subvert immunoregulatory processes to protect themselves from destruction by T cells, to the detriment of patients. A growing body of evidence supports the hypothesis that specialized subsets of dendritic cells expressing indoleamine 2,3 dioxygenase (IDO), which catalyzes oxidative catabolism of tryptophan, play critical roles in regulation of T cell-mediated immune responses. IDO-dependent T cell suppression by dendritic cells suggests that biochemical changes due to tryptophan catabolism have profound effects on T cell proliferation, differentiation, effector functions, and viability. This has critical implications for immunotherapeutic manipulations designed for patients with cancer and chronic infectious diseases. In this review, I focus on dendritic cells that can express IDO, and which acquire potent T cell regulatory functions as a consequence.

  19. Lignans from Carthamus tinctorius suppress tryptophan breakdown via indoleamine 2,3-dioxygenase.

    PubMed

    Kuehnl, Susanne; Schroecksnadel, Sebastian; Temml, Veronika; Gostner, Johanna M; Schennach, Harald; Schuster, Daniela; Schwaiger, Stefan; Rollinger, Judith M; Fuchs, Dietmar; Stuppner, Hermann

    2013-10-15

    Seed extracts of Carthamus tinctorius L. (Asteraceae), safflower, have been traditionally used to treat coronary disease, thrombotic disorders, and menstrual problems but also against cancer and depression. A possible effect of C. tinctorius compounds on tryptophan-degrading activity of enzyme indoleamine 2,3-dioxygenase (IDO) could explain many of its activities. To test for an effect of C. tinctorius extracts and isolated compounds on cytokine-induced IDO activity in immunocompetent cells in vitro methanol and ethylacetate seed extracts were prepared from cold pressed seed cakes of C. tinctorius and three lignan derivatives, trachelogenin, arctigenin and matairesinol were isolated. The influence on tryptophan breakdown was investigated in peripheral blood mononuclear cells (PBMCs). Effects were compared to neopterin production in the same cellular assay. Both seed extracts suppressed tryptophan breakdown in stimulated PBMC. The three structurally closely related isolates exerted differing suppressive activity on PBMC: arctigenin (IC50 26.5μM) and trachelogenin (IC50 of 57.4μM) showed higher activity than matairesinol (IC50 >200μM) to inhibit tryptophan breakdown. Effects on neopterin production were similar albeit generally less strong. Data show an immunosuppressive property of compounds which slows down IDO activity. The in vitro results support the view that some of the anti-inflammatory, anticancer and antidepressant properties of C. tinctorius lignans might relate to their suppressive influence on tryptophan breakdown.

  20. Therapeutic antibody targeting of indoleamine-2,3-dioxygenase (IDO2) inhibits autoimmune arthritis.

    PubMed

    Merlo, Lauren M F; Grabler, Samantha; DuHadaway, James B; Pigott, Elizabeth; Manley, Kaylend; Prendergast, George C; Laury-Kleintop, Lisa D; Mandik-Nayak, Laura

    2017-02-20

    Rheumatoid arthritis (RA) is a debilitating inflammatory autoimmune disease with no known cure. Recently, we identified the immunomodulatory enzyme indoleamine-2,3-dioxygenase 2 (IDO2) as an essential mediator of autoreactive B and T cell responses driving RA. However, therapeutically targeting IDO2 has been challenging given the lack of small molecules that specifically inhibit IDO2 without also affecting the closely related IDO1. In this study, we develop a novel monoclonal antibody (mAb)-based approach to therapeutically target IDO2. Treatment with IDO2-specific mAb alleviated arthritis in two independent preclinical arthritis models, reducing autoreactive T and B cell activation and recapitulating the strong anti-arthritic effect of genetic IDO2 deficiency. Mechanistic investigations identified FcγRIIb as necessary for mAb internalization, allowing targeting of an intracellular antigen traditionally considered inaccessible to mAb therapy. Taken together, our results offer preclinical proof of concept for antibody-mediated targeting of IDO2 as a new therapeutic strategy to treat RA and other autoantibody-mediated diseases.

  1. Insights into DNA hydroxymethylation in the honeybee from in-depth analyses of TET dioxygenase.

    PubMed

    Wojciechowski, Marek; Rafalski, Dominik; Kucharski, Robert; Misztal, Katarzyna; Maleszka, Joanna; Bochtler, Matthias; Maleszka, Ryszard

    2014-08-01

    In mammals, a family of TET enzymes producing oxidized forms of 5-methylcytosine (5mC) plays an important role in modulating DNA demethylation dynamics. In contrast, nothing is known about the function of a single TET orthologue present in invertebrates. Here, we show that the honeybee TET (AmTET) catalytic domain has dioxygenase activity and converts 5mC to 5-hydroxymethylcytosine (5hmC) in a HEK293T cell assay. In vivo, the levels of 5hmC are condition-dependent and relatively low, but in testes and ovaries 5hmC is present at approximately 7-10% of the total level of 5mC, which is comparable to that reported for certain mammalian cells types. AmTET is alternatively spliced and highly expressed throughout development and in adult tissues with the highest expression found in adult brains. Our findings reveal an additional level of flexible genomic modifications in the honeybee that may be important for the selection of multiple pathways controlling contrasting phenotypic outcomes in this species. In a broader context, our study extends the current, mammalian-centred attention to TET-driven DNA hydroxymethylation to an easily manageable organism with attractive and unique biology.

  2. Keratin degradation by dermatophytes relies on cysteine dioxygenase and a sulfite efflux pump.

    PubMed

    Grumbt, Maria; Monod, Michel; Yamada, Tsuyoshi; Hertweck, Christian; Kunert, Jiri; Staib, Peter

    2013-06-01

    Millions of people suffer from superficial infections caused by dermatophytes. Intriguingly, these filamentous fungi exclusively infect keratin-rich host structures such as hair, nails, and skin. Keratin is a hard, compact protein, and its utilization by dermatophytes for growth has long been discussed as a major virulence attribute. Here, we provide strong support for the hypothesis that keratin degradation is facilitated by the secretion of the reducing agent sulfite, which can cleave keratin-stabilizing cystine bonds. We discovered that sulfite is produced by dermatophytes from environmental cysteine, which at elevated concentrations is toxic for microbes and humans. We found that sulfite formation from cysteine relies on the key enzyme cysteine dioxygenase Cdo1. Sulfite secretion is supported by the sulfite efflux pump Ssu1. Targeted mutagenesis proved that dermatophyte mutants in either Cdo1 or Ssu1 were highly growth-sensitive to cysteine, and mutants in Ssu1 were specifically sensitive to sulfite. Most notably, dermatophyte mutants in Cdo1 and Ssu1 were specifically growth-defective on hair and nails. As keratin is rich in cysteine, our identified mechanism of cysteine conversion and sulfite efflux supports both cysteine and sulfite tolerance per se and progression of keratin degradation. These in vitro findings have implications for dermatophyte infection pathogenesis.

  3. Substrate Recognition and Catalysis by the Cofactor-Independent Dioxygenase DpgC+

    SciTech Connect

    Fielding,E.; Widboom, P.; Bruner, S.

    2007-01-01

    The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3, 5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.

  4. Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds.

    PubMed

    Stacey, Minviluz G; Cahoon, Rebecca E; Nguyen, Hanh T; Cui, Yaya; Sato, Shirley; Nguyen, Cuong T; Phoka, Nongnat; Clark, Kerry M; Liang, Yan; Forrester, Joe; Batek, Josef; Do, Phat Tien; Sleper, David A; Clemente, Thomas E; Cahoon, Edgar B; Stacey, Gary

    2016-11-01

    Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits.

  5. Diversity and distribution of catechol 2, 3-dioxygenase genes in surface sediments of the Bohai Sea.

    PubMed

    He, Peiqing; Li, Li; Liu, Jihua; Bai, Yazhi; Fang, Xisheng

    2016-05-01

    Catechol 2, 3-dioxygenase (C23O) is the key enzyme for aerobic aromatic degradation. Based on clone libraries and quantitative real-time polymerase chain reaction, we characterized diversity and distribution patterns of C23O genes in surface sediments of the Bohai Sea. The results showed that sediments of the Bohai Sea were dominated by genes related to C23O subfamily I.2.A. The samples from wastewater discharge area (DG) and aquaculture farm (KL) showed distinct composition of C23O genes when compared to the samples from Bohai Bay (BH), and total organic carbon was a crucial determinant accounted for the composition variation. C6BH12-38 and C2BH2-35 displayed the highest gene copies and highest ratios to the 16S rRNA genes in KL, and they might prefer biologically labile aromatic hydrocarbons via aquaculture inputs. Meanwhile, C7BH3-48 showed the highest gene copies and highest ratios to the 16S rRNA genes in DG, and this could be selective effect of organic loadings from wastewater discharge. An evident increase in C6BH12-38 and C7BH3-48 gene copies and reduction in diversity of C23O genes in DG and KL indicated composition perturbations of C23O genes and potential loss in functional redundancy. We suggest that ecological habitat and trophic specificity could shape the distribution of C23O genes in the Bohai Sea sediments.

  6. Suicide inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols

    SciTech Connect

    Bartels, I.; Knackmuss, H.J.; Reineke, W.

    1984-03-01

    The inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-chloro- and 3-fluorocatechol and the iron-chelating agent Tiron (catechol-3,5-disulfonate) was studied. Whereas inactivation by Tiron is an oxygen-independent and mostly reversible process, inactivation by the 3-halocatechols was only observed in the presence of oxygen and was largely irreversible. The rate constants for inactivation (K/sub 2/) were 1.62 x 10/sup -3/ sec/sup -1/ for 3-chlorocatechol and 2.38 x 10/sup -3/ sec/sup -1/ for 3-fluorocatechol. The inhibitor constants (K/sub i/) were 23 ..mu..M for 3-chlorocatechol and 17 ..mu..M for 3-fluorocatechol. The kinetic data for 3-fluorocatechol could only be obtained in the presence of 2-mercaptoethanol. Besides inactivated enzyme, some 2-hydroxyhexa-2,4-dienoic acid as the actual suicide product of meta-cleavage. A side product of 3-fluorocatechol cleavage is a yellow compound with the spectral characteristics of a 2-hydroxy-6-oxohexa-2,4-dienoci acid indicating 1,6-cleavage. Rates of inactivation by 3-fluorocatechol were reduced in the presence of superoxide dismutase, catalase, formate, and mannitol, which implies that superoxide anion, hydrogen peroxide, and hydroxyl radical exhibit additional inactivation. 64 references.

  7. Brain indoleamine 2,3-dioxygenase contributes to the comorbidity of pain and depression

    PubMed Central

    Kim, Hyangin; Chen, Lucy; Lim, Grewo; Sung, Backil; Wang, Shuxing; McCabe, Michael F.; Rusanescu, Gabriel; Yang, Liling; Tian, Yinghong; Mao, Jianren

    2012-01-01

    Pain and depression are frequently comorbid disorders, but the mechanism underlying this association is unknown. Here, we report that brain indoleamine 2,3-dioxygenase 1 (IDO1), a rate-limiting enzyme in tryptophan metabolism, plays a key role in this comorbidity. We found that chronic pain in rats induced depressive behavior and IDO1 upregulation in the bilateral hippocampus. Upregulation of IDO1 resulted in the increased kynurenine/tryptophan ratio and decreased serotonin/tryptophan ratio in the bilateral hippocampus. We observed elevated plasma IDO activity in patients with both pain and depression, as well as in rats with anhedonia induced by chronic social stress. Intra-hippocampal administration of IL-6 in rats, in addition to in vitro experiments, demonstrated that IL-6 induces IDO1 expression through the JAK/STAT pathway. Further, either Ido1 gene knockout or pharmacological inhibition of hippocampal IDO1 activity attenuated both nociceptive and depressive behavior. These results reveal an IDO1-mediated regulatory mechanism underlying the comorbidity of pain and depression and suggest a new strategy for the concurrent treatment of both conditions via modulation of brain IDO1 activity. PMID:22751107

  8. Substrate recognition and catalysis by the cofactor-independent dioxygenase DpgC.

    PubMed

    Fielding, Elisha N; Widboom, Paul F; Bruner, Steven D

    2007-12-11

    The enzyme DpgC belongs to a small class of oxygenases not dependent on accessory cofactors for activity. DpgC is in the biosynthetic pathway for the nonproteinogenic amino acid 3,5-dihydroxyphenylglycine in actinomycetes bacteria responsible for the production of the vancomycin/teicoplanin family of antibiotic natural products. The X-ray structure of DpgC [Widboom, P. W., Fielding, E. N., Liu, Y., and Bruner, S. D. (2007) Nature 447, 342-345] confirmed the absence of cofactors and defined a novel hydrophobic dioxygen binding pocket adjacent to a bound substrate analogue. In this paper, the role specific amino acids play in substrate recognition and catalysis is examined through biochemical and structural characterization of site-specific enzyme mutations and alternate substrates. The results establish the importance of three amino acids, Arg254, Glu299, and Glu189, in the chemistry of DpgC. Arg254 and Glu189 join to form a specific contact with one of the phenolic hydroxyls of the substrate, and this interaction plays a key role in both substrate recognition and catalysis. The X-ray crystal structure of Arg254Lys was determined to address the role this residue plays in the chemistry. In addition, characterization of alternate substrate analogues demonstrates the presence and position of phenol groups are necessary for both enzyme recognition and downstream oxidation chemistry. Overall, this work defines the mechanism of substrate recognition and specificity by the cofactor-independent dioxygenase DpgC.

  9. Intracerebroventricular administration of lipopolysaccharide induces indoleamine-2,3-dioxygenase-dependent depression-like behaviors

    PubMed Central

    2013-01-01

    Background Activation of the tryptophan degrading enzyme indoleamine-2,3-dioxygenase 1 (IDO1) is associated with the development of behavioral signs of depression. Systemic immune challenge induces IDO1 in both the periphery and the brain, leading to increased circulating and brain concentrations of kynurenines. However, whether IDO1 activity within the brain is necessary for the manifestation of depression-like behavior of mice following a central immune challenge remains to be elucidated. Methods We investigated the role of brain IDO1 in mediating depression-like behavior of mice in response to intracerebroventricular injection of saline or lipopolysaccharide (LPS, 10 ng). Results LPS increased the duration of immobility in the tail suspension test and decreased preference for a sucrose solution. These effects were associated with an activation of central but not peripheral IDO1, as LPS increased brain kynurenine but had no effect on plasma concentrations of kynurenine. Interestingly, genetic deletion or pharmacological inhibition of IDO1, using 1-methyl-tryptophan, abrogated the reduction in sucrose preference induced by intracerebroventricular LPS. 1-Methyl-tryptophan also blocked the LPS-induced increase in duration of immobility during the tail suspension test. Conclusions These data indicate that activation of brain IDO1 is sufficient to induce depression-like behaviors of mice in response to central LPS. PMID:23866724

  10. Salmonella overcomes tumor immune tolerance by inhibition of tumor indoleamine 2, 3-dioxygenase 1 expression.

    PubMed

    Kuan, Yu-Diao; Lee, Che-Hsin

    2016-01-05

    Over the past decades, Salmonella has been proven capable of inhibiting tumor growth. It can specifically target tumors and due to its facultative anaerobic property, can be more penetrative than other drug therapies. However, the molecular mechanism by which Salmonella inhibits tumor growth is still incompletely known. The antitumor therapeutic effect mediated by Salmonella is associated with an inflammatory immune response at the tumor site and a T cell-dependent immune response. Many tumors have been proven to have a high expression of indoleamine 2, 3-dioxygenase 1 (IDO), which is a rate-limiting enzyme that catalyzes tryptophan to kynurenine, thus causing immune tolerance within the tumor microenvironment. With decreased expression of IDO, increased immune response can be observed, which might be helpful when developing cancer immunotherapy. The expression of IDO was decreased after tumor cells were infected with Salmonella. In addition, Western blot analysis showed that the expression levels of phospho-protein kinase B (P-AKT), phospho-mammalian targets of rapamycin (P-mTOR), and phospho-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were decreased after Salmonella infection. In conclusion, our results indicate that Salmonella inhibits IDO expression and plays a crucial role in anti-tumor therapy, which might be a promising strategy combined with other cancer treatments.

  11. Distribution and prediction of catalytic domains in 2-oxoglutarate dependent dioxygenases

    PubMed Central

    2012-01-01

    Background The 2-oxoglutarate dependent superfamily is a diverse group of non-haem dioxygenases, and is present in prokaryotes, eukaryotes, and archaea. The enzymes differ in substrate preference and reaction chemistry, a factor that precludes their classification by homology studies and electronic annotation schemes alone. In this work, I propose and explore the rationale of using substrates to classify structurally similar alpha-ketoglutarate dependent enzymes. Findings Differential catalysis in phylogenetic clades of 2-OG dependent enzymes, is determined by the interactions of a subset of active-site amino acids. Identifying these with existing computational methods is challenging and not feasible for all proteins. A clustering protocol based on validated mechanisms of catalysis of known molecules, in tandem with group specific hidden markov model profiles is able to differentiate and sequester these enzymes. Access to this repository is by a web server that compares user defined unknown sequences to these pre-defined profiles and outputs a list of predicted catalytic domains. The server is free and is accessible at the following URL ( http://comp-biol.theacms.in/H2OGpred.html). Conclusions The proposed stratification is a novel attempt at classifying and predicting 2-oxoglutarate dependent function. In addition, the server will provide researchers with a tool to compare their data to a comprehensive list of HMM profiles of catalytic domains. This work, will aid efforts by investigators to screen and characterize putative 2-OG dependent sequences. The profile database will be updated at regular intervals. PMID:22862831

  12. Lignans from Carthamus tinctorius suppress tryptophan breakdown via indoleamine 2,3-dioxygenase

    PubMed Central

    Kuehnl, Susanne; Schroecksnadel, Sebastian; Temml, Veronika; Gostner, Johanna M.; Schennach, Harald; Schuster, Daniela; Schwaiger, Stefan; Rollinger, Judith M.; Fuchs, Dietmar; Stuppner, Hermann

    2013-01-01

    Seed extracts of Carthamus tinctorius L. (Asteraceae), safflower, have been traditionally used to treat coronary disease, thrombotic disorders, and menstrual problems but also against cancer and depression. A possible effect of C. tinctorius compounds on tryptophan-degrading activity of enzyme indoleamine 2,3-dioxygenase (IDO) could explain many of its activities. To test for an effect of C. tinctorius extracts and isolated compounds on cytokine-induced IDO activity in immunocompetent cells in vitro methanol and ethylacetate seed extracts were prepared from cold pressed seed cakes of C. tinctorius and three lignan derivatives, trachelogenin, arctigenin and matairesinol were isolated. The influence on tryptophan breakdown was investigated in peripheral blood mononuclear cells (PBMCs). Effects were compared to neopterin production in the same cellular assay. Both seed extracts suppressed tryptophan breakdown in stimulated PBMC. The three structurally closely related isolates exerted differing suppressive activity on PBMC: arctigenin (IC50 26.5 μM) and trachelogenin (IC50 of 57.4 μM) showed higher activity than matairesinol (IC50 >200 μM) to inhibit tryptophan breakdown. Effects on neopterin production were similar albeit generally less strong. Data show an immunosuppressive property of compounds which slows down IDO activity. The in vitro results support the view that some of the anti-inflammatory, anti-cancer and antidepressant properties of C. tinctorius lignans might relate to their suppressive influence on tryptophan breakdown. PMID:23867649

  13. Analysis of phylogenetic and functional diverge in plant nine-cis epoxycarotenoid dioxygenase gene family.

    PubMed

    Priya, R; Siva, Ramamoorthy

    2015-07-01

    During different environmental stress conditions, plant growth is regulated by the hormone abscisic acid (an apocarotenoid). In the biosynthesis of abscisic acid, the oxidative cleavage of cis-epoxycarotenoid catalyzed by 9-cis-epoxycarotenoid dioxygenase (NCED) is the crucial step. The NCED genes were isolated in numerous plant species and those genes were phylogenetically investigated to understand the evolution of NCED genes in various plant lineages comprising lycophyte, gymnosperm, dicot and monocot. A total of 93 genes were obtained from 48 plant species to statistically estimate their sequence conservation and functional divergence. Selaginella moellendorffii appeared to be evolutionarily distinct from those of the angiosperms, insisting the substantial influence of natural selection pressure on NCED genes. Further, using exon-intron structure analysis, the gene structures of NCED were found to be conserved across some species. In addition, the substitution rate ratio of non-synonymous (Ka) versus synonymous (Ks) mutations using the Bayesian inference approach, depicted the critical amino acid residues for functional divergence. A significant functional divergence was found between some subgroups through the co-efficient of type-I functional divergence. Our results suggest that the evolution of NCED genes occurred by duplication, diversification and exon intron loss events. The site-specific profile and functional diverge analysis revealed NCED genes might facilitate the tissue-specific functional divergence in NCED sub-families, that could combat different environmental stress conditions aiding plant survival.

  14. Induction of 9-cis-epoxycarotenoid dioxygenase in Arabidopsis thaliana seeds enhances seed dormancy.

    PubMed

    Martínez-Andújar, Cristina; Ordiz, M Isabel; Huang, Zhonglian; Nonogaki, Mariko; Beachy, Roger N; Nonogaki, Hiroyuki

    2011-10-11

    Full understanding of mechanisms that control seed dormancy and germination remains elusive. Whereas it has been proposed that translational control plays a predominant role in germination, other studies suggest the importance of specific gene expression patterns in imbibed seeds. Transgenic plants were developed to permit conditional expression of a gene encoding 9-cis-epoxycarotenoid dioxygenase 6 (NCED6), a rate-limiting enzyme in abscisic acid (ABA) biosynthesis, using the ecdysone receptor-based plant gene switch system and the ligand methoxyfenozide. Induction of NCED6 during imbibition increased ABA levels more than 20-fold and was sufficient to prevent seed germination. Germination suppression was prevented by fluridone, an inhibitor of ABA biosynthesis. In another study, induction of the NCED6 gene in transgenic seeds of nondormant mutants tt3 and tt4 reestablished seed dormancy. Furthermore, inducing expression of NCED6 during seed development suppressed vivipary, precocious germination of developing seeds. These results indicate that expression of a hormone metabolism gene in seeds can be a sole determinant of dormancy. This study opens the possibility of developing a robust technology to suppress or promote seed germination through engineering pathways of hormone metabolism.

  15. Identification of Homogentisate Dioxygenase as a Target for Vitamin E Biofortification in Oilseeds1[OPEN

    PubMed Central

    Nguyen, Hanh T.; Cui, Yaya; Sato, Shirley; Clark, Kerry M.; Liang, Yan; Forrester, Joe; Batek, Josef; Do, Phat Tien; Sleper, David A.

    2016-01-01

    Soybean (Glycine max) is a major plant source of protein and oil and produces important secondary metabolites beneficial for human health. As a tool for gene function discovery and improvement of this important crop, a mutant population was generated using fast neutron irradiation. Visual screening of mutagenized seeds identified a mutant line, designated MO12, which produced brown seeds as opposed to the yellow seeds produced by the unmodified Williams 82 parental cultivar. Using forward genetic methods combined with comparative genome hybridization analysis, we were able to establish that deletion of the GmHGO1 gene is the genetic basis of the brown seeded phenotype exhibited by the MO12 mutant line. GmHGO1 encodes a homogentisate dioxygenase (HGO), which catalyzes the committed enzymatic step in homogentisate catabolism. This report describes to our knowledge the first functional characterization of a plant HGO gene, defects of which are linked to the human genetic disease alkaptonuria. We show that reduced homogentisate catabolism in a soybean HGO mutant is an effective strategy for enhancing the production of lipid-soluble antioxidants such as vitamin E, as well as tolerance to herbicides that target pathways associated with homogentisate metabolism. Furthermore, this work demonstrates the utility of fast neutron mutagenesis in identifying novel genes that contribute to soybean agronomic traits. PMID:27660165

  16. D2HGDH regulates alpha-ketoglutarate levels and dioxygenase function by modulating IDH2

    PubMed Central

    Lin, An-Ping; Abbas, Saman; Kim, Sang-Woo; Ortega, Manoela; Bouamar, Hakim; Escobedo, Yissela; Varadarajan, Prakash; Qin, Yuejuan; Sudderth, Jessica; Schulz, Eduard; Deutsch, Alexander; Mohan, Sumitra; Ulz, Peter; Neumeister, Peter; Rakheja, Dinesh; Gao, Xiaoli; Hinck, Andrew; Weintraub, Susan T.; DeBerardinis, Ralph J.; Sill, Heinz; Dahia, Patricia L. M.; Aguiar, Ricardo C. T.

    2015-01-01

    Isocitrate dehydrogenases (IDH) convert isocitrate to alpha-ketoglutarate (α-KG). In cancer, mutant IDH1/2 reduces α-KG to D2-hydroxyglutarate (D2-HG) disrupting α-KG-dependent dioxygenases. However, the physiological relevance of controlling the interconversion of D2-HG into α-KG, mediated by D2-hydroxyglutarate dehydrogenase (D2HGDH), remains obscure. Here we show that wild-type D2HGDH elevates α-KG levels, influencing histone and DNA methylation, and HIF1α hydroxylation. Conversely, the D2HGDH mutants that we find in diffuse large B-cell lymphoma are enzymatically inert. D2-HG is a low-abundance metabolite, but we show that it can meaningfully elevate α-KG levels by positively modulating mitochondrial IDH activity and inducing IDH2 expression. Accordingly, genetic depletion of IDH2 abrogates D2HGDH effects, whereas ectopic IDH2 rescues D2HGDH-deficient cells. Our data link D2HGDH to cancer and describe an additional role for the enzyme: the regulation of IDH2 activity and α-KG-mediated epigenetic remodelling. These data further expose the intricacies of mitochondrial metabolism and inform on the pathogenesis of D2HGDH-deficient diseases. PMID:26178471

  17. Indoleamine 2,3-dioxygenase attenuates inhibitor development in gene-therapy-treated hemophilia A mice.

    PubMed

    Liu, L; Liu, H; Mah, C; Fletcher, B S

    2009-06-01

    A serious impediment to gene and protein replacement therapy in hemophilia A is the development of inhibitors. Mechanisms responsible for inhibitor development include T-cell-dependent adaptive immune responses and the CD28-B7 signaling pathway that eventually leads to the formation of antibodies directed against factor VIII (FVIII). Indoleamine 2,3-dioxygenase (IDO) is a potent immunosuppressive enzyme that can inhibit T-cell responses and induce T-cell apoptosis by regulation of tryptophan metabolism. Kynurenine, one of the metabolites of tryptophan, has been implicated as an immune modulator. Here we hypothesize that co-delivery of the genes for FVIII and IDO can attenuate inhibitor formation. Using transposon-based gene delivery, we observed long-term therapeutic FVIII expression and significantly reduced inhibitor titers when the genes were co-delivered. Co-expression of FVIII and IDO in the liver was associated with increased plasma kynurenine levels, an inhibition of T-cell infiltration and increased apoptosis of T cells within the liver. These experiments suggest that modulation of tryptophan catabolism through IDO expression provides a novel strategy to reduce inhibitor development in hemophilia gene/protein therapy.

  18. Ormosil gels doped with engineered catechol 1,2 dioxygenases for chlorocatechol bioremediation.

    PubMed

    Micalella, Chiara; Caglio, Raffaella; Mozzarelli, Andrea; Valetti, Francesca; Pessione, Enrica; Giunta, Carlo; Bruno, Stefano

    2014-01-01

    Enzymes entrapped in wet, nanoporous silica gel have great potential as bioreactors for bioremediation because of their improved thermal, chemical, and mechanical stability with respect to enzymes in solution. The B isozyme of catechol 1,2 dioxygenase from Acinetobacter radioresistens and its mutants of Leu69 and Ala72, designed for an increased reactivity toward the environmental pollutant chlorocatechols, were encapsulated using alkoxysilanes and alkyl alkoxysilanes as precursors in varying proportions. Encapsulation of the mutants in a hydrophobic tetramethoxysilane/dimethoxydimethylsilane-based matrix yielded a remarkable 10- to 12-fold enhancement in reactivity toward chlorocatechols. These gels also showed a fivefold increase in relative reactivity toward chlorocatechols with respect to the natural substrate catechol, thus compensating for their relatively low activity for these substrates in solution. The encapsulated enzyme, unlike the enzyme in solution, proved resilient in assays carried out in urban wastewater and bacteria-contaminated solutions mimicking environmentally relevant conditions. Overall, the combination of a structure-based rational design of enzyme mutants, and the selection of a suitable encapsulation material, proved to be a powerful approach for the production and optimization of a potential bioremediation device, with increased activity and resistance toward bacterial degradation.

  19. Inhibition of allogeneic T-cell response by Kupffer cells expressing indoleamine 2,3-dioxygenase

    PubMed Central

    Yan, Mao-Lin; Wang, Yao-Dong; Tian, Yi-Feng; Lai, Zhi-De; Yan, Lv-Nan

    2010-01-01

    AIM: To explore the possibility and mechanism of inhibiting allogeneic T-cell responses by Kupffer cells (KC) pretreated with interferon-γ (IFN-γ) in vitro. METHODS: The expressions of indoleamine 2,3-dioxygenase (IDO) mRNA and FasL mRNA in KC pretreated with IFN-γ were studied with real-time polymerase chain reaction (PCR). The catabolism of tryptophan by IDO from KC was analyzed by high performance liquid chromatography. Allogeneic T-cell response was used to confirm the inhibition of KC in vitro. The proliferation of lymphocytes was detected using [3H] thymidine incorporation. Cell cycle and lymphocyte apoptosis were evaluated by flow cytometric assay. RESULTS: Real-time PCR revealed IDO mRNA and FasL mRNA expressions in KC pretreated with IFN-γ, and IDO catabolic effect was confirmed by a decrease in tryptophan and increase in kynurenine concentration. KC expressing IDO and FasL in BABL/c mice acquired the ability to suppress the proliferation of T-cells from C57BL/6, which could be blocked by addition of 1-methyl-tryptophan and anti-FasL antibody. KC expressing IDO could induce allogeneic T-cell apoptosis. CONCLUSION: In addition to Fas/FasL pathway, IDO may be another mechanism for KC to induce immune tolerance. PMID:20128035

  20. Indoleamine 2,3-dioxygenase 1 (IDO1) inhibitors activate the aryl hydrocarbon receptor.

    PubMed

    Moyer, Benjamin J; Rojas, Itzel Y; Murray, Iain A; Lee, Seokwon; Hazlett, Haley F; Perdew, Gary H; Tomlinson, Craig R

    2017-03-20

    Indoleamine 2,3-dioxygenase 1 (IDO1) plays a key role in the immune system by regulating tryptophan levels and T cell differentiation. Several tumor types overexpress IDO1 to avoid immune surveillance making IDO1 of interest as a target for therapeutic intervention. As a result, several IDO1 inhibitors are currently being tested in clinical trials for cancer treatment as well as several other diseases. Many of the IDO1 inhibitors in clinical trials naturally bear structural similarities to the IDO1 substrate tryptophan, as such, they fulfill many of the structural and functional criteria as potential AHR ligands. Using mouse and human cell-based luciferase gene reporter assays, qPCR confirmation experiments, and CYP1A1 enzyme activity assays, we report that some of the promising clinical IDO1 inhibitors also act as agonists for the aryl hydrocarbon receptor (AHR), best known for its roles in xenobiotic metabolism and as another key regulator of the immune response. The dual role as IDO antagonist and AHR agonist for many of these IDO target drugs should be considered for full interrogation of their biological mechanisms and clinical outcomes.

  1. Salmonella overcomes tumor immune tolerance by inhibition of tumor indoleamine 2, 3-dioxygenase 1 expression

    PubMed Central

    Kuan, Yu-Diao; Lee, Che-Hsin

    2016-01-01

    Over the past decades, Salmonella has been proven capable of inhibiting tumor growth. It can specifically target tumors and due to its facultative anaerobic property, can be more penetrative than other drug therapies. However, the molecular mechanism by which Salmonella inhibits tumor growth is still incompletely known. The antitumor therapeutic effect mediated by Salmonella is associated with an inflammatory immune response at the tumor site and a T cell-dependent immune response. Many tumors have been proven to have a high expression of indoleamine 2, 3-dioxygenase 1 (IDO), which is a rate-limiting enzyme that catalyzes tryptophan to kynurenine, thus causing immune tolerance within the tumor microenvironment. With decreased expression of IDO, increased immune response can be observed, which might be helpful when developing cancer immunotherapy. The expression of IDO was decreased after tumor cells were infected with Salmonella. In addition, Western blot analysis showed that the expression levels of phospho-protein kinase B (P-AKT), phospho-mammalian targets of rapamycin (P-mTOR), and phospho-p70 ribosomal s6 kinase (P-p70s6K) in tumor cells were decreased after Salmonella infection. In conclusion, our results indicate that Salmonella inhibits IDO expression and plays a crucial role in anti-tumor therapy, which might be a promising strategy combined with other cancer treatments. PMID:26517244

  2. Carotenoid cleavage dioxygenase4 is a negative regulator of β-carotene content in Arabidopsis seeds.

    PubMed

    Gonzalez-Jorge, Sabrina; Ha, Sun-Hwa; Magallanes-Lundback, Maria; Gilliland, Laura Ullrich; Zhou, Ailing; Lipka, Alexander E; Nguyen, Yen-Nhu; Angelovici, Ruthie; Lin, Haining; Cepela, Jason; Little, Holly; Buell, C Robin; Gore, Michael A; Dellapenna, Dean

    2013-12-01

    Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID cleavage dioxygenase4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops.

  3. Identification and characterization of a novel three-component phenazine-1-carboxylic acid 1, 2-dioxygenase in Sphingomonas wittichii DP58.

    PubMed

    Zhao, Qiang; Hu, Hong-Bo; Wang, Wei; Huang, Xian-Qing; Zhang, Xue-Hong

    2017-02-10

    Phenazine-1-carboxylic acid, the main component of shenqinmycin, is widely used in southern China for the prevention of rice sheath blight. However, the fate of phenazine-1-carboxylic acid in soil remains uncertain. S. wittichii DP58 can use phenazine-1-carboxylic acid as its sole carbon and nitrogen sources for growth. In this study, dioxygenase-encoding genes, pcaA1A2, were found using transcriptome analysis to be highly upregulated upon phenazine-1-carboxylic acid biodegradation. PcaA1 shares 68% amino acid sequence identity with the large oxygenase subunit of anthranilate 1, 2-dioxygenase from R. maanshanensis DSM 44675. The dioxygenase was co-expressed in E. coli with its adjacent reductase encoding gene, pcaA3 and ferredoxin encoding gene, pcaA4, and showed phenazine-1-carboxylic acid consumption. The dioxygenase-, ferredoxin- and reductase-encoding genes were expressed in P. putida KT2440 or E. coli BL21, and the three recombinant proteins were purified. A phenazine-1-carboxylic acid conversion capability occurred in vitro only when all three components were present. However, P. putida KT2440 transformed with pcaA1A2 obtained phenazine-1-carboxylic acid degradation ability, suggesting that phenazine-1-carboxylic acid 1, 2-dioxygenase has low specificities for its ferredoxin and reductase. This was verified by replacing PcaA3 with RedA2 in the in vitro enzyme assay. HPLC/MS and NMR analysis showed that phenazine-1-carboxylic acid was converted to 1, 2-dihydroxy phenazine through decarboxylation and hydroxylation, indicating that PcaA1A2A3A4 constitutes the initial phenazine-1-carboxylic acid 1, 2-dioxygenase. This study fills a gap in our understanding of the biodegradation of phenazine-1-carboxylic acid and illustrates a new dioxygenase for decarboxylation.Importance Phenazine-1-carboxylic acid is widely used in southern China as a key fungicide to prevent rice sheath blight. However, the degradation characteristics of phenazine-1-carboxylic acid and the

  4. The two enantiospecific dichlorprop/alpha-ketoglutarate-dioxygenases from Delftia acidovorans MC1--protein and sequence data of RdpA and SdpA.

    PubMed

    Westendorf, Anne; Benndorf, Dirk; Müller, Roland H; Babel, Wolfgang

    2002-01-01

    Two alpha-ketoglutarate-dependent dioxygenases carrying enantiospecific activity for the etherolytic cleavage of racemic phenoxypropionate herbicides [(RS)-2-(2,4-dichlorophenoxy)propionate and (RS)-2-(4-chloro-2-methylphenoxy)propionate] from Delftia acidovorans MC1 were characterized with respect to protein and sequence data. The (S)-phenoxypropionate/alpha-ketoglutarate-dioxygenase (SdpA) appeared as a monomeric enzyme with a molecular weight of 32 kDa in the presence of SDS. N-terminal sequences revealed relationship to alpha-ketoglutarate-dependent taurine dioxygenase (TauD) and to 2,4-dichlorophenoxyacetate/alpha-ketoglutarate-dioxygenase (TfdA). The (R)-phenoxypropionate/alpha-ketoglutarate-dioxygenase (RdpA) referred to 36 kDa in the presence of SDS and to 108 kDa under native conditions. Internal sequences of fragments obtained after digestion made evident relationship to TfdA and TauD. Two-dimensional electrophoretic separation resulted in the resolution of up to 3 individual spots with almost identical molecular weights but different isoelectric points with both RdpA and SdpA. The structural differences of these isoenzyme forms are not yet clear.

  5. Re-evaluation of dioxygenase gene phylogeny for the development and validation of a quantitative assay for environmental aromatic hydrocarbon degraders

    PubMed Central

    Meynet, Paola; Head, Ian M.; Werner, David; Davenport, Russell J.

    2015-01-01

    Rieske non-heme iron oxygenases enzymes have been widely studied, as they catalyse essential reactions initiating the bacterial degradation of organic compounds, for instance aromatic hydrocarbons. The genes encoding these enzymes offer a potential target for studying aromatic hydrocarbon-degrading organisms in the environment. However, previously reported primer sets that target dioxygenase gene sequences or the common conserved Rieske centre of aromatics dioxygenases have limited specificity and/or target non-dioxygenase genes. In this work, an extensive database of dioxygenase α-subunit gene sequences was constructed, and primer sets targeting the conserved Rieske centre were developed. The high specificity of the primers was confirmed by polymerase chain reaction analysis, agarose gel electrophoresis and sequencing. Quantitative polymerase chain reaction (qPCR) assays were also developed and optimized, following MIQE guidelines (Minimum Information for Publication of Quantitative Real-Time PCR Experiments). Comparison of the qPCR quantification of dioxygenases in spiked sediment samples and in pure cultures demonstrated an underestimation of the Ct value, and the requirement for a correction factor at gene abundances below 108 gene copies per g of sediment. Externally validated qPCR provides a valuable tool to monitor aromatic hydrocarbon degrader population abundances at contaminated sites. PMID:25944871

  6. Characterizing the Promiscuity of LigAB, a Lignin Catabolite Degrading Extradiol Dioxygenase from Sphingomonas paucimobilis SYK-6

    PubMed Central

    Barry, Kevin P.; Taylor, Erika A.

    2014-01-01

    LigAB from Sphingomonas paucimobilis SYK-6 is the only structurally characterized dioxygenase of the largely uncharacterized superfamily of Type II extradiol dioxygenases (EDO). This enzyme catalyzes the oxidative ring-opening of protocatechuate (3,4-dihydroxybenzoic acid or PCA) in a pathway allowing the degradation of lignin derived aromatic compounds (LDACs). LigAB has also been shown to utilize two other LDACs from the same metabolic pathway as substrates, gallate, and 3-O-methyl gallate; however, kcat/KM had not been reported for any of these compounds. In order to assess the catalytic efficiency and get insights into the observed promiscuity of this enzyme, steady-state kinetic analyses were performed for LigAB with these and a library of related compounds. The dioxygenation of PCA by LigAB was highly efficient, with a kcat of 51 s−1 and a kcat/KM of 4.26 × 106 M−1s−1. LigAB demonstrated the ability to use a variety of catecholic molecules as substrates beyond the previously identified gallate and 3-O-methyl gallate, including 3,4-dihydroxybenzamide, homoprotocatechuate, catechol, and 3,4-dihydroxybenzonitrile. Interestingly, 3,4-dihydroxybenzamide (DHBAm) behaves in a manner similar to that of the preferred benzoic acid substrates, with a kcat/Km value only ~4-fold lower than that for gallate and ~10-fold higher than that for 3-O-methyl gallate. All of these most active substrates demonstrate mechanistic inactivation of LigAB. Additionally, DHBAm exhibits potent product inhibition that leads to an inactive enzyme, being more highly deactivating at lower substrate concentration, a phenomena that, to our knowledge, has not been reported for another dioxygenase substrate/product pair. These results provide valuable catalytic insight into the reactions catalyzed by LigAB and make it the first Type II EDO that is fully characterized both structurally and kinetically. PMID:23977959

  7. Isolation and characterization of Pseudomonas putida PpF1 mutants defective in the toluene dioxygenase enzyme system.

    PubMed Central

    Finette, B A; Subramanian, V; Gibson, D T

    1984-01-01

    Pseudomonas putida PpF1 degraded toluene via a dihydrodiol pathway to tricarboxylic acid cycle intermediates. The initial reaction was catalyzed by a multicomponent enzyme, toluene dioxygenase, which oxidized toluene to (+)-cis-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). The enzyme consisted of three protein components: NADH-ferredoxintol oxidoreductase (reductasetol), ferredoxintol, and a terminal oxygenase which is an iron-sulfur protein (ISPtol). Mutants blocked in each of these components were isolated after mutagenesis with nitrosoguanidine. Mutants occurred as colony morphology variants when grown in the presence of toluene on indicator plates containing agar, mineral salts, a growth-supporting nutrient (arginine), 2,3,5-triphenyltetrazolium chloride (TTC), and Nitro Blue Tetrazolium (NBT). Under these conditions, wild-type colonies appeared large and red as a result of TTC reduction. Colonies of reductasetol mutants were white or white with a light blue center, ferredoxintol strains were light blue with a dark blue center, and strains that lacked ISPtol gave dark blue colonies. Blue color differences in the mutant colonies were due to variations in the extent of NBT reduction. Strains lacking all three components appeared white. Toluene dioxygenase mutants were characterized by assaying toluene dioxygenase activity in crude cell extracts which were complemented with purified preparations of each protein component. Between 40 and 60% of the putative mutants selected from the NBT-TTC indicator plates were unable to grow with toluene as the sole source of carbon and energy. This method should prove extremely useful in isolating mutants in other multicomponent oxygenase enzyme systems. Images PMID:6501223

  8. Bioinspired copper(I) complexes that exhibit monooxygenase and catechol dioxygenase activity.

    PubMed

    Arnold, Aline; Metzinger, Ramona; Limberg, Christian

    2015-01-12

    New tripodal ligand L2 featuring three different pyridyl/imidazolyl-based N-donor units at a bridgehead C atom, from which one of the imidazolyl units is separated by a phenylene linker, was synthesized and investigated with regards to copper(I) complexation. The resulting complex [(L2)Cu]OTf (2(OTf)), the known complex [(L1)Cu]OTf (1(OTf); L1 differs from L2 in that it lacks the phenylene spacer) and [(L3)Cu]OTf (3(OTf)), prepared from a known chiral, tripodal, N-donor ligand featuring pyridyl, pyrazolyl, and imidazolyl donors, were tested as catalysts for the oxidation of sodium 2,4-di-tert-butylphenolate (NaDTBP) with O2. Indeed, they mediated NaDTBP oxidation to give mainly the corresponding catecholate and quinone (Q). None of the complexes 1(OTf), 2(OTf), and 3(OTf) is superior to the others, as yields were comparable and, if the presence of protons is guaranteed by concomitant addition of the phenol DTBP, the oxidation can also be performed catalytically. For all complexes stoichiometric oxidations under certain conditions (concentrated solutions, high NaDTBP content) were found to also generate products typical for metal-mediated intradiol cleavage of the catecholate with O2. As shown representatively for 1(OTf) this dioxygenation sets in at a later stage of the reaction. Initially a copper species responsible for the monooxygenation must form from 1(OTf)/NaDTBP/O2, and only thereafter is the copper species responsible for dioxygenation formed and consumes Q as substrate. Hence, under these circumstances complexes 1(OTf)-3(OTf) show both monooxygenase and catechol dioxygenase activity.

  9. Isolation and characterization of carotenoid cleavage dioxygenase 4 genes from different citrus species.

    PubMed

    Zheng, Xiongjie; Xie, Zongzhou; Zhu, Kaijie; Xu, Qiang; Deng, Xiuxin; Pan, Zhiyong

    2015-08-01

    In plants, the carotenoid cleavage dioxygenase 4 (CCD4) could target on plastoglobules and cleave specific carotenoids, producing apocarotenoids and volatile compounds. These compounds are important for color and aroma formation in fruits and flowers. In this study, five CCD4 gene members (CCD4a, b, c, d, and e) were investigated in different citrus species including mandarin, pummelo, and sweet orange. Sequence analysis showed that the CCD4 genes from all the species examined exhibited extensive allelic variability (including SNPs and frame-shift mutations). Furthermore, the distribution of the CCD4 allelic mutation sites supported our previous hypothesis that the sweet orange originated from the hybridization of mandarin and pummelo. A derived cleaved amplified polymorphic sequence (dCAPs) marker was then successfully developed based on the allelic polymorphism of CCD4c, providing an ideal molecular marker for studying the genetic relationship between citrus species. Quantitative RT-PCR analysis identified differential expression patterns for the CCD4 genes in tissues/organs, and CCD4b was shown to have a high-level expression in citrus fruit flavedos (especially those with a deep orange-reddish color). HPLC-based detection of a key component (i.e., β-citraurin) for orange-reddish flavedo formation in different citrus revealed a positive correlation between CCD4b expression levels and the presence of β-citraurin, suggesting that CCD4b may be responsible for β-citraurin biosynthesis in flavedo. In summary, this study not only reinforced the anticipated roles of CCD4 genes in flavedo color formation in citrus, but also provided new information about gene expression patterns, allelic polymorphism characteristics, and sequence variability for this gene subfamily.

  10. Mutation and polymorphism analysis of the human homogentisate 1, 2-dioxygenase gene in alkaptonuria patients.

    PubMed

    Beltrán-Valero de Bernabé, D; Granadino, B; Chiarelli, I; Porfirio, B; Mayatepek, E; Aquaron, R; Moore, M M; Festen, J J; Sanmartí, R; Peñalva, M A; de Córdoba, S R

    1998-04-01

    Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles.

  11. Mutation and polymorphism analysis of the human homogentisate 1, 2-dioxygenase gene in alkaptonuria patients.

    PubMed Central

    Beltrán-Valero de Bernabé, D; Granadino, B; Chiarelli, I; Porfirio, B; Mayatepek, E; Aquaron, R; Moore, M M; Festen, J J; Sanmartí, R; Peñalva, M A; de Córdoba, S R

    1998-01-01

    Alkaptonuria (AKU), a rare hereditary disorder of phenylalanine and tyrosine catabolism, was the first disease to be interpreted as an inborn error of metabolism. AKU patients are deficient for homogentisate 1,2 dioxygenase (HGO); this deficiency causes homogentisic aciduria, ochronosis, and arthritis. We cloned the human HGO gene and characterized two loss-of-function mutations, P230S and V300G, in the HGO gene in AKU patients. Here we report haplotype and mutational analysis of the HGO gene in 29 novel AKU chromosomes. We identified 12 novel mutations: 8 (E42A, W97G, D153G, S189I, I216T, R225H, F227S, and M368V) missense mutations that result in amino acid substitutions at positions conserved in HGO in different species, 1 (F10fs) frameshift mutation, 2 intronic mutations (IVS9-56G-->A, IVS9-17G-->A), and 1 splice-site mutation (IVS5+1G-->T). We also report characterization of five polymorphic sites in HGO and describe the haplotypic associations of alleles at these sites in normal and AKU chromosomes. One of these sites, HGO-3, is a variable dinucleotide repeat; IVS2+35T/A, IVS5+25T/C, and IVS6+46C/A are intronic sites at which single nucleotide substitutions (dimorphisms) have been detected; and c407T/A is a relatively frequent nucleotide substitution in the coding sequence, exon 4, resulting in an amino acid change (H80Q). These data provide insight into the origin and evolution of the various AKU alleles. PMID:9529363

  12. The molecular basis for adaptive evolution in novel extradiol dioxygenases retrieved from the metagenome.

    PubMed

    Suenaga, Hikaru; Mizuta, Shiori; Miyazaki, Kentaro

    2009-09-01

    Extradiol dioxygenase (EDO) catalyzes metal-dependent ring cleavage of catecholic substrates. We previously screened a metagenomic library of activated sludge used to treat industrial wastewater contaminated with phenols and cyanide to identify 43 EDO genes. Here, we have characterized the enzymes belonging to novel I.2.G, I.3.M and I.3.N subfamilies. The I.3.M and I.3.N EDOs were Fe(II) dependent and preferred bicyclic substrates, whereas the I.2.G EDOs were Mn(II) dependent, preferred monocyclic substrates and had the highest affinity for catechol reported thus far. The I.2.G EDOs were more tolerant against heat (60 degrees C for 1 h) and chemical inhibitors (H(2)O(2) and NaCN) than I.3.M and I.3.N EDOs. Considering the dominance of the I.2.G EDOs over all retrieved EDOs (20 of 43 clones) and the presence of cyanide in the environment, this high affinity for substrate and structural robustness should provide survival advantages to host microorganisms. The 20 I.2.G EDOs were classified into six groups based on the amino acid sequence of the predicted ancestor, 1A1. Enzymes were chosen from each group and characterized. Two descendents, 1D2 and 5B2, each had a k(cat)/K(M) approximately twofold higher than that of 1A1 and reduced thermal stability, suggesting that descendents of 1A1 have adapted evolutionarily by a trade-off of inherent stability for increased activity.

  13. Substrate and Cofactor Range Differences of Two Cysteine Dioxygenases from Ralstonia eutropha H16

    PubMed Central

    Wenning, Leonie; Stöveken, Nadine; Wübbeler, Jan Hendrik

    2015-01-01

    Cysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3′-thiodipropionic acid (TDP) and 3,3′-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs. PMID:26590284

  14. Isotope effects of enzymatic dioxygenation of nitrobenzene and 2-nitrotoluene by nitrobenzene dioxygenase.

    PubMed

    Pati, Sarah G; Kohler, Hans-Peter E; Bolotin, Jakov; Parales, Rebecca E; Hofstetter, Thomas B

    2014-09-16

    Oxygenation of aromatic rings is a frequent initial step in the biodegradation of persistent contaminants, and the accompanying isotope fractionation is increasingly used to assess the extent of transformation in the environment. Here, we systematically investigated the dioxygenation of two nitroaromatic compounds (nitrobenzene and 2-nitrotoluene) by nitrobenzene dioxygenase (NBDO) to obtain insights into the factors governing its C, H, and N isotope fractionation. Experiments were carried out at different levels of biological complexity from whole bacterial cells to pure enzyme. C, H, and N isotope enrichment factors and kinetic isotope effects (KIEs) were derived from the compound-specific isotope analysis of nitroarenes, whereas C isotope fractionation was also quantified in the oxygenated reaction products. Dioxygenation of nitrobenzene to catechol and 2-nitrotoluene to 3-methylcatechol showed large C isotope enrichment factors, ϵC, of -4.1 ± 0.2‰ and -2.5 ± 0.2‰, respectively, and was observed consistently in the substrates and dioxygenation products. ϵH- and ϵN-values were smaller, that is -5.7 ± 1.3‰ and -1.0 ± 0.3‰, respectively. C isotope fractionation was also identical in experiments with whole bacterial cells and pure enzymes. The corresponding (13)C-KIEs for the dioxygenation of nitrobenzene and 2-nitrotoluene were 1.025 ± 0.001 and 1.018 ± 0.001 and suggest a moderate substrate specificity. Our study illustrates that dioxygenation of nitroaromatic contaminants exhibits a large C isotope fractionation, which is not masked by substrate transport and uptake processes and larger than dioxygenation of other aromatic hydrocarbons.

  15. Tet methylcytosine dioxygenase 2 inhibits atherosclerosis via upregulation of autophagy in ApoE−/− mice

    PubMed Central

    Peng, Juan; Yang, Qin; Li, A-Fang; Li, Rong-Qing; Wang, Zuo; Liu, Lu-Shan; Ren, Zhong; Zheng, Xi-Long; Tang, Xiao-Qing; Li, Guo-Hua; Tang, Zhi-Han; Jiang, Zhi-Sheng; Wei, Dang-Heng

    2016-01-01

    Tet methylcytosine dioxygenase 2 (TET2) mediates the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). The loss of TET2 is associated with advanced atherosclerotic lesions. Our previous study showed that TET2 improves endothelial cell function by enhancing endothelial cell autophagy. Accordingly, this study determined the role of TET2 in atherosclerosis and potential mechanisms. In ApoE−/− mice fed high-fat diet, TET2 overexpression markedly decreased atherosclerotic lesions with uniformly increased level of 5hmC and decreased level of 5mC in genomic DNA. TET2 overexpression also promoted autophagy and downregulated inflammation factors, such as vascular cell adhesion molecule 1, intercellular adhesion molecule 1, monocyte chemotactic protein 1, and interleukin-1. Consistently, TET2 knockdown with small hairpin RNA (shRNA) in ApoE−/− mice decreased 5hmC and increased 5mC levels in atherosclerotic lesions. Meanwhile, autophagy was inhibited and atherosclerotic lesions progressed with an unstable lesion phenotype characterized by large lipid core, macrophage accumulation, and upregulated inflammation factor expression. Experiments with the cultured endothelial cells revealed that oxidized low-density lipoprotein (ox-LDL) inhibited endothelial cell autophagy. TET2 shRNA strengthened impaired autophagy and autophagic flux in the ox-LDL-treated endothelial cells. TET2 overexpression reversed these effects by decreasing the methylation level of the Beclin 1 promoter, which contributed to the downregulation of inflammation factors. Overall, we identified that TET2 was downregulated during the pathogenesis of atherosclerosis. The downregulation of TET2 promotes the methylation of the Beclin 1 promoter, leading to endothelial cell autophagy, impaired autophagic flux, and inflammatory factor upregulation. Upregulation of TET2 may be a novel therapeutic strategy for treating atherosclerosis. PMID:27821816

  16. Expression of indoleamine 2,3-dioxygenase in a murine model of Aspergillus fumigatus keratitis

    PubMed Central

    Jiang, Nan; Zhao, Gui-Qiu; Lin, Jing; Hu, Li-Ting; Che, Cheng-Ye; Li, Cui; Wang, Qian; Xu, Qiang; Zhang, Jie; Peng, Xu-Dong

    2016-01-01

    AIM To observe the presence and expression of indoleamine 2,3-dioxygenase (IDO) during the corneal immunity to Aspergillus fumigatus (A. fumigatus) in the murine models. METHODS The murine model of fungal keratitis was established by smearing with colonies of A. fumigatus after scraping central epithelium of cornea and covering with contact lenses in C57BL/6 mice. The mice were randomly divided into control group, sham group and A. fumigatus keratitis group. The cornea was monitored daily using a slit lamp and recorded disease score after infection. Corneal lesion was detected by immunofluorescence staining. IDO mRNA and protein were also detected by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. RESULTS The disease score and slit lamp photography indicated that disease severity was consistent with corneal inflammation in the murine models, and the disease scores in A. fumigatus keratitis group were obviously higher than those in the sham group. By immunofluorescence staining, IDO was mainly localized in corneal epithelium and stroma in the murine corneal tissues with A. fumigatus keratitis. Compared with the sham group, IDO mRNA expression was significantly enhanced in corneal epithelium infected by A. fumigatus. Furthermore, IDO protein expression detected by Western blot was in accord with transcript levels of IDO mRNA measured by qRT-PCR. IDO protein expression was enhanced after A. fumigatus infection compared with the sham group. CONCLUSION IDO is detected in corneal epithelium and stroma locally, which indicates IDO takes part in the pathogenesis of A. fumigatus keratitis and plays a key role in immune regulation at the early stage. PMID:27162718

  17. Structural basis for the enantiospecificities of R- and S-specific phenoxypropionate/α-ketoglutarate dioxygenases

    PubMed Central

    Müller, Tina A.; Zavodszky, Maria I.; Feig, Michael; Kuhn, Leslie A.; Hausinger, Robert P.

    2006-01-01

    (R)- and (S)-dichlorprop/α-ketoglutarate dioxygenases (RdpA and SdpA) catalyze the oxidative cleavage of 2-(2,4-dichlorophenoxy)propanoic acid (dichlorprop) and 2-(4-chloro-2-methyl-phenoxy)propanoic acid (mecoprop) to form pyruvate plus the corresponding phenol concurrent with the conversion of α-ketoglutarate (αKG) to succinate plus CO2. RdpA and SdpA are strictly enantiospecific, converting only the (R) or the (S) enantiomer, respectively. Homology models were generated for both enzymes on the basis of the structure of the related enzyme TauD (PDB code 1OS7). Docking was used to predict the orientation of the appropriate mecoprop enantiomer in each protein, and the predictions were tested by characterizing the activities of site-directed variants of the enzymes. Mutant proteins that changed at residues predicted to interact with (R)- or (S)-mecoprop exhibited significantly reduced activity, often accompanied by increased Km values, consistent with roles for these residues in substrate binding. Four of the designed SdpA variants were (slightly) active with (R)-mecoprop. The results of the kinetic investigations are consistent with the identification of key interactions in the structural models and demonstrate that enantiospecificity is coordinated by the interactions of a number of residues in RdpA and SdpA. Most significantly, residues Phe171 in RdpA and Glu69 in SdpA apparently act by hindering the binding of the wrong enantiomer more than the correct one, as judged by the observed decreases in Km when these side chains are replaced by Ala. PMID:16731970

  18. Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes.

    PubMed

    Wright, Terry R; Shan, Guomin; Walsh, Terence A; Lira, Justin M; Cui, Cory; Song, Ping; Zhuang, Meibao; Arnold, Nicole L; Lin, Gaofeng; Yau, Kerrm; Russell, Sean M; Cicchillo, Robert M; Peterson, Mark A; Simpson, David M; Zhou, Ning; Ponsamuel, Jayakumar; Zhang, Zhanyuan

    2010-11-23

    Engineered glyphosate resistance is the most widely adopted genetically modified trait in agriculture, gaining widespread acceptance by providing a simple robust weed control system. However, extensive and sustained use of glyphosate as a sole weed control mechanism has led to field selection for glyphosate-resistant weeds and has induced significant population shifts to weeds with inherent tolerance to glyphosate. Additional weed control mechanisms that can complement glyphosate-resistant crops are, therefore, urgently needed. 2,4-dichlorophenoxyacetic acid (2,4-D) is an effective low-cost, broad-spectrum herbicide that controls many of the weeds developing resistance to glyphosate. We investigated the substrate preferences of bacterial aryloxyalkanoate dioxygenase enzymes (AADs) that can effectively degrade 2,4-D and have found that some members of this class can act on other widely used herbicides in addition to their activity on 2,4-D. AAD-1 cleaves the aryloxyphenoxypropionate family of grass-active herbicides, and AAD-12 acts on pyridyloxyacetate auxin herbicides such as triclopyr and fluroxypyr. Maize plants transformed with an AAD-1 gene showed robust crop resistance to aryloxyphenoxypropionate herbicides over four generations and were also not injured by 2,4-D applications at any growth stage. Arabidopsis plants expressing AAD-12 were resistant to 2,4-D as well as triclopyr and fluroxypyr, and transgenic soybean plants expressing AAD-12 maintained field resistance to 2,4-D over five generations. These results show that single AAD transgenes can provide simultaneous resistance to a broad repertoire of agronomically important classes of herbicides, including 2,4-D, with utility in both monocot and dicot crops. These transgenes can help preserve the productivity and environmental benefits of herbicide-resistant crops.

  19. Robust crop resistance to broadleaf and grass herbicides provided by aryloxyalkanoate dioxygenase transgenes

    PubMed Central

    Wright, Terry R.; Shan, Guomin; Walsh, Terence A.; Lira, Justin M.; Cui, Cory; Song, Ping; Zhuang, Meibao; Arnold, Nicole L.; Lin, Gaofeng; Yau, Kerrm; Russell, Sean M.; Cicchillo, Robert M.; Peterson, Mark A.; Simpson, David M.; Zhou, Ning; Ponsamuel, Jayakumar; Zhang, Zhanyuan

    2010-01-01

    Engineered glyphosate resistance is the most widely adopted genetically modified trait in agriculture, gaining widespread acceptance by providing a simple robust weed control system. However, extensive and sustained use of glyphosate as a sole weed control mechanism has led to field selection for glyphosate-resistant weeds and has induced significant population shifts to weeds with inherent tolerance to glyphosate. Additional weed control mechanisms that can complement glyphosate-resistant crops are, therefore, urgently needed. 2,4-dichlorophenoxyacetic acid (2,4-D) is an effective low-cost, broad-spectrum herbicide that controls many of the weeds developing resistance to glyphosate. We investigated the substrate preferences of bacterial aryloxyalkanoate dioxygenase enzymes (AADs) that can effectively degrade 2,4-D and have found that some members of this class can act on other widely used herbicides in addition to their activity on 2,4-D. AAD-1 cleaves the aryloxyphenoxypropionate family of grass-active herbicides, and AAD-12 acts on pyridyloxyacetate auxin herbicides such as triclopyr and fluroxypyr. Maize plants transformed with an AAD-1 gene showed robust crop resistance to aryloxyphenoxypropionate herbicides over four generations and were also not injured by 2,4-D applications at any growth stage. Arabidopsis plants expressing AAD-12 were resistant to 2,4-D as well as triclopyr and fluroxypyr, and transgenic soybean plants expressing AAD-12 maintained field resistance to 2,4-D over five generations. These results show that single AAD transgenes can provide simultaneous resistance to a broad repertoire of agronomically important classes of herbicides, including 2,4-D, with utility in both monocot and dicot crops. These transgenes can help preserve the productivity and environmental benefits of herbicide-resistant crops. PMID:21059954

  20. Expression of α-DIOXYGENASE 1 in tomato and Arabidopsis contributes to plant defenses against aphids.

    PubMed

    Avila, Carlos Augusto; Arevalo-Soliz, Lirio Milenka; Lorence, Argelia; Goggin, Fiona L

    2013-08-01

    Plant α-dioxygenases (α-DOX) are fatty acid-hydroperoxidases that contribute to the synthesis of oxylipins, a diverse group of compounds primarily generated through oxidation of linoleic (LA) and linolenic acid (LNA). Oxylipins are implicated in plant signaling against biotic and abiotic stresses. We report here that the potato aphid (Macrosiphum euphorbiae) induces Slα-DOX1 but not Slα-DOX2 expression in tomato (Solanum lycopersicum). Slα-DOX1 upregulation by aphids does not require either jasmonic acid (JA) or salicylic acid (SA) accumulation, since tomato mutants deficient in JA (spr2, acx1) or SA accumulation (NahG) still show Slα-DOX1 induction. Virus-induced gene silencing of Slα-DOX1 enhanced aphid population growth in wild-type (WT) plants, revealing that Slα-DOX1 contributes to basal resistance to aphids. Moreover, an even higher percent increase in aphid numbers occurred when Slα-DOX1 was silenced in spr2, a mutant line characterized by elevated LA levels, decreased LNA, and enhanced aphid resistance as compared with WT. These results suggest that aphid reproduction is influenced by oxylipins synthesized from LA by Slα-DOX1. In agreement with our experiments in tomato, two independent α-dox1 T-DNA insertion mutant lines in Arabidopsis thaliana also showed increased susceptibility to the green peach aphid (Myzus persicae), indicating that the role α-DOX is conserved in other plant-aphid interactions.

  1. Indoleamine 2,3-dioxygenase pathways of pathgenic inflammation and immune escape in cancer

    PubMed Central

    Prendergast, George C.; Smith, Courtney; Thomas, Sunil; Mandik-Nayak, Laura; Laury-Kleintop, Lisa; Metz, Richard; Muller, Alexander J.

    2014-01-01

    Genetic and pharmacological studies of indoleamine 2,3-dioxygenase (IDO) have established this tryptophan catabolic enzyme as a central driver of malignant development and progression. IDO acts in tumor, stromal and immune cells to support pathogenic inflammatory processes that engender immune tolerance to tumor antigens. The multifaceted effects of IDO activation in cancer include the suppression of T and NK cells, the generation and activation of T regulatory cells (Treg) and myeloid-derived suppressor cells (MDSC), and the promotion of tumor angiogenesis. Mechanistic investigations have defined the aryl hydrocarbon receptor AhR, the master metabolic regulator mTORC1 and the stress kinase Gcn2 as key effector signaling elements for IDO, which also exerts a non-catalytic role in TGF-β signaling. Small molecule inhibitors of IDO exhibit anticancer activity and cooperate with immunotherapy, radiotherapy or chemotherapy to trigger rapid regression of aggressive tumors otherwise resistant to treatment. Notably, the dramatic antitumor activity of certain targeted therapeutics such as imatinib (Gleevec) in GIST has been traced in part to IDO downregulation. Further, antitumor responses to immune checkpoint inhibitors can be heightened safely by a clinical lead inhibitor of the IDO pathway that relieves IDO-mediated suppression of mTORC1 in T cells. In this personal perspective on IDO as a nodal mediator of pathogenic inflammation and immune escape in cancer, we provide a conceptual foundation for the clinical development of IDO inhibitors as a novel class of immunomodulators with broad application in the treatment of advanced human cancer. PMID:24711084

  2. A 2-Oxoglutarate-Dependent Dioxygenase Mediates the Biosynthesis of Glucoraphasatin in Radish1[OPEN

    PubMed Central

    Kitashiba, Hiroyasu; Li, Feng; Fukino, Nobuko; Ohara, Takayoshi; Nishio, Takeshi; Ishida, Masahiko

    2017-01-01

    Glucosinolates (GSLs) are secondary metabolites whose degradation products confer intrinsic flavors and aromas to Brassicaceae vegetables. Several structures of GSLs are known in the Brassicaceae, and the biosynthetic pathway and regulatory networks have been elucidated in Arabidopsis (Arabidopsis thaliana). GSLs are precursors of chemical defense substances against herbivorous pests. Specific GSLs can act as feeding blockers or stimulants, depending on the pest species. Natural selection has led to diversity in the GSL composition even within individual species. However, in radish (Raphanus sativus), glucoraphasatin (4-methylthio-3-butenyl glucosinolate) accounts for more than 90% of the total GSLs, and little compositional variation is observed. Because glucoraphasatin is not contained in other members of the Brassicaceae, like Arabidopsis and cabbage (Brassica oleracea), the biosynthetic pathways for glucoraphasatin remain unclear. In this report, we identified and characterized a gene encoding GLUCORAPHASATIN SYNTHASE 1 (GRS1) by genetic mapping using a mutant that genetically lacks glucoraphasatin. Transgenic Arabidopsis, which overexpressed GRS1 cDNA, accumulated glucoraphasatin in the leaves. GRS1 encodes a 2-oxoglutarate-dependent dioxygenase, and it is abundantly expressed in the leaf. To further investigate the biosynthesis and transportation of GSLs in radish, we grafted a grs1 plant onto a wild-type plant. The grafting experiment revealed a leaf-to-root long-distance glucoraphasatin transport system in radish and showed that the composition of GSLs differed among the organs. Based on these observations, we propose a characteristic biosynthesis pathway for glucoraphasatin in radish. Our results should be useful in metabolite engineering for breeding of high-value vegetables. PMID:28100450

  3. Stepwise conversion of flavonoids by engineered dioxygenases and dehydrogenase: Characterization of novel biotransformation products.

    PubMed

    Overwin, Heike; González, Myriam; Méndez, Valentina; Cárdenas, Franco; Seeger, Michael; Hofer, Bernd

    2015-12-01

    Flavonoids are a large group of plant secondary metabolites that exert various biological and pharmacological effects. In this context, the generation of derivatives is of considerable interest. The introduction of hydroxy groups is of particular relevance, as they are known to be involved in many of the biological interactions and furthermore enable additional modifications, such as glycosylations. Bacterial aryl-hydroxylating dioxygenases (ARHDOs) have proven to be very useful for the conversion of aromatic structures into versatile building blocks for different kinds of derivatizations. Such enzymes have been used with varying success for the oxidation of flavonoids. In order to find better ARHDOs for the hydroxylation of such substrates, we carried out biotransformation trials with a collection of hybrid ARHDOs of different origin, using resting cells of recombinant strains. This identified enzymes able to transform all of the flavonoids examined, typically in yields above 50%. It also showed that moderately reactive substituents of flavonoids, such as hydroxy or amino groups, can lead to spontaneous follow-up reactions with the dienediol structures generated by dioxygenation. A report of flavanone epoxidation, a reaction never before observed to be catalyzed by an ARHDO, is challenged by our results. All ARHDOs examined converted this substrate into a dehydrogenase-transformable dihydrodiol. All dihydrodiols obtained by dioxygenation of the examined flavonoids were successfully re-aromatized into catechols by a bacterial dehydrogenase. These metabolites were usually stable. However, the catechols formed from flavanone and 2'-hydroxy-chalcone, respectively, were interconvertible under mild conditions. Altogether, we isolated and characterized 13 compounds that have not previously been described. The biotransformations reported here give access to novel flavonoid derivatives that may be applied for biological screens as well as for further modification, such as

  4. Dioxygenase-encoding AtDAO1 gene controls IAA oxidation and homeostasis in Arabidopsis

    PubMed Central

    Porco, Silvana; Pěnčík, Aleš; Rashed, Afaf; Voß, Ute; Casanova-Sáez, Rubén; Bishopp, Anthony; Golebiowska, Agata; Swarup, Ranjan; Swarup, Kamal; Peňáková, Pavlína; Novák, Ondřej; Staswick, Paul; Hedden, Peter; Phillips, Andrew L.; Vissenberg, Kris

    2016-01-01

    Auxin represents a key signal in plants, regulating almost every aspect of their growth and development. Major breakthroughs have been made dissecting the molecular basis of auxin transport, perception, and response. In contrast, how plants control the metabolism and homeostasis of the major form of auxin in plants, indole-3-acetic acid (IAA), remains unclear. In this paper, we initially describe the function of the Arabidopsis thaliana gene DIOXYGENASE FOR AUXIN OXIDATION 1 (AtDAO1). Transcriptional and translational reporter lines revealed that AtDAO1 encodes a highly root-expressed, cytoplasmically localized IAA oxidase. Stable isotope-labeled IAA feeding studies of loss and gain of function AtDAO1 lines showed that this oxidase represents the major regulator of auxin degradation to 2-oxoindole-3-acetic acid (oxIAA) in Arabidopsis. Surprisingly, AtDAO1 loss and gain of function lines exhibited relatively subtle auxin-related phenotypes, such as altered root hair length. Metabolite profiling of mutant lines revealed that disrupting AtDAO1 regulation resulted in major changes in steady-state levels of oxIAA and IAA conjugates but not IAA. Hence, IAA conjugation and catabolism seem to regulate auxin levels in Arabidopsis in a highly redundant manner. We observed that transcripts of AtDOA1 IAA oxidase and GH3 IAA-conjugating enzymes are auxin-inducible, providing a molecular basis for their observed functional redundancy. We conclude that the AtDAO1 gene plays a key role regulating auxin homeostasis in Arabidopsis, acting in concert with GH3 genes, to maintain auxin concentration at optimal levels for plant growth and development. PMID:27651491

  5. Evolution and diversity of the 2-oxoglutarate-dependent dioxygenase superfamily in plants.

    PubMed

    Kawai, Yosuke; Ono, Eiichiro; Mizutani, Masaharu

    2014-04-01

    The 2-oxoglutarate-dependent dioxygenase (2OGD) superfamily is the second largest enzyme family in the plant genome, and its members are involved in various oxygenation/hydroxylation reactions. Despite their biochemical significance in metabolism, a systematic analysis of plant 2OGDs remains to be accomplished. We present a phylogenetic classification of 479 2OGDs in six plant models, ranging from green algae to angiosperms. These were classified into three classes - DOXA, DOXB and DOXC - based on amino acid sequence similarity. The DOXA class includes plant homologs of Escherichia coli AlkB, which is a prototype of 2OGD involved in the oxidative demethylation of alkylated nucleic acids and histones. The DOXB class is conserved across all plant taxa and is involved in proline 4-hydroxylation in cell wall protein synthesis. The DOXC class is involved in specialized metabolism of various phytochemicals, including phytohormones and flavonoids. The vast majority of 2OGDs from land plants were classified into the DOXC class, but only seven from Chlamydomonas, suggesting that this class has diversified during land plant evolution. Phylogenetic analysis assigned DOXC-class 2OGDs to 57 phylogenetic clades. 2OGD genes involved in gibberellin biosynthesis were conserved among vascular plants, and those involved in flavonoid and ethylene biosynthesis were shared among seed plants. Several angiosperm-specific clades were found to be involved in various lineage-specific specialized metabolisms, but 31 of the 57 DOXC-class clades were only found in a single species. Therefore, the evolution and diversification of DOXC-class 2OGDs is partly responsible for the diversity and complexity of specialized metabolites in land plants.

  6. Homology modeling, simulation and molecular docking studies of catechol-2, 3-Dioxygenase from Burkholderia cepacia: Involved in degradation of Petroleum hydrocarbons

    PubMed Central

    Ajao, AT; Kannan, M; Yakubu, SE; VJ, Umoh; JB, Ameh

    2012-01-01

    Catechol 2, 3-dioxygenase is present in several types of bacteria and undergoes degradation of environmental pollutants through an important key biochemical pathways. Specifically, this enzyme cleaves aromatic rings of several environmental pollutants such as toluene, xylene, naphthalene and biphenyl derivatives. Hence, the importance of Catechol 2, 3-dioxygenase and its role in the degradation of environmental pollutants made us to predict the three-dimensional structure of Catechol 2, 3-dioxygenase from Burkholderia cepacia. The 10ns molecular dynamics simulation was carried out to check the stability of the modeled Catechol 2, 3- dioxygenase. The results show that the model was energetically stable, and it attains their equilibrium within 2000 ps of production MD run. The docking of various petroleum hydrocarbons into the Catechol 2,3-dioxygenase reveals that the benzene, O-xylene, Toluene, Fluorene, Naphthalene, Carbazol, Pyrene, Dibenzothiophene, Anthracene, Phenanthrene, Biphenyl makes strong hydrogen bond and Van der waals interaction with the active site residues of H150, L152, W198, H206, H220, H252, I254, T255, Y261, E271, L276 and F309. Free energy of binding and estimated inhibition constant of these compounds demonstrates that they are energetically stable in their binding cavity. Chrysene shows positive energy of binding in the active site atom of Fe. Except Pyrene all the substrates made close contact with Fe atom by the distance ranges from 1.67 to 2.43 Å. In addition to that, the above mentioned substrate except pyrene all other made π-π stacking interaction with H252 by the distance ranges from 3.40 to 3.90 Å. All these docking results reveal that, except Chrysene all other substrate has good free energy of binding to hold enough in the active site and makes strong VdW interaction with Catechol-2,3-dioxygenase. These results suggest that, the enzyme is capable of catalyzing the above-mentioned substrate. PMID:23144539

  7. Selection for Growth on 3-Nitrotoluene by 2-Nitrotoluene-Utilizing Acidovorax sp. Strain JS42 Identifies Nitroarene Dioxygenases with Altered Specificities

    PubMed Central

    Mahan, Kristina M.; Penrod, Joseph T.; Ju, Kou-San; Al Kass, Natascia; Tan, Watumesa A.; Truong, Richard; Parales, Juanito V.

    2014-01-01

    Acidovorax sp. strain JS42 uses 2-nitrotoluene as a sole source of carbon and energy. The first enzyme of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase, adds both atoms of molecular oxygen to 2-nitrotoluene, forming nitrite and 3-methylcatechol. All three mononitrotoluene isomers serve as substrates for 2-nitrotoluene dioxygenase, but strain JS42 is unable to grow on 3- or 4-nitrotoluene. Using both long- and short-term selections, we obtained spontaneous mutants of strain JS42 that grew on 3-nitrotoluene. All of the strains obtained by short-term selection had mutations in the gene encoding the α subunit of 2-nitrotoluene dioxygenase that changed isoleucine 204 at the active site to valine. Those strains obtained by long-term selections had mutations that changed the same residue to valine, alanine, or threonine or changed the alanine at position 405, which is just outside the active site, to glycine. All of these changes altered the regiospecificity of the enzymes with 3-nitrotoluene such that 4-methylcatechol was the primary product rather than 3-methylcatechol. Kinetic analyses indicated that the evolved enzymes had enhanced affinities for 3-nitrotoluene and were more catalytically efficient with 3-nitrotoluene than the wild-type enzyme. In contrast, the corresponding amino acid substitutions in the closely related enzyme nitrobenzene 1,2-dioxygenase were detrimental to enzyme activity. When cloned genes encoding the evolved dioxygenases were introduced into a JS42 mutant lacking a functional dioxygenase, the strains acquired the ability to grow on 3-nitrotoluene but with significantly longer doubling times than the evolved strains, suggesting that additional beneficial mutations occurred elsewhere in the genome. PMID:25344236

  8. Five-coordinate M(II)-semiquinonate (M = Fe, Mn, Co) complexes: reactivity models of the catechol dioxygenases.

    PubMed

    Wang, Peng; Yap, Glenn P A; Riordan, Charles G

    2014-06-04

    A series of five-coordinate M(II)-semiquinonate (M = Fe, Mn, Co) complexes were synthesized and characterized, including the first example of a mononuclear Fe(II)-semiquinonate. Intermediates were observed in the reactions of M(II)-phenSQ (M = Fe, Co) with O2. Evidence for the relevance of these intermediates to the intradiol catechol dioxygenases was obtained by characterization of the oxidized semiquinone-derived product, muconic anhydride, resulting from the reaction of [PhTt(tBu)]Co(II)(3,5-DBSQ) with O2.

  9. Stereospecific hydroxylation of indan by Escherichia coli containing the cloned toluene dioxygenase genes from Pseudomonas putida F1

    SciTech Connect

    Brand, J.M.; Cruden, D.L.; Zylstra, G.J.; Gibson, D.T. )

    1992-10-01

    Escheria coli JM109(pDTG601), containing the todC1C2BA genes encoding toluene dioxygenase from Pseudomonas putida F1, oxides indan to (-)-(1R)-indanol (83{percent} R) and trans-1,3-indandiol. Under similar conditions, P.putida F39/D oxidizes indan to (-)-(1R)-indanol (96{percent}R), 1-indanone, and trans-1,3-indandiol. The differences in the enantiomeric composition of the 1-indanols formed by the two organisms are due to the presence of a 1-indanol dehydrogenase in P.putida F39/D that preferentially oxidizes (+)-(1S)-indanol.

  10. Synthetic, Spectroscopic and DFT Studies of Iron Complexes with Iminobenzo(semi)quinone Ligands: Implications for o-Aminophenol Dioxygenases

    PubMed Central

    Bittner, Michael M.; Kraus, David; Lindeman, Sergey V.; Popescu, Codrina V.; Fiedler, Adam T.

    2014-01-01

    The oxidative C-C bond cleavage of o-aminophenols by nonheme Fe dioxygenases is a critical step in both human metabolism (the kynurenine pathway) and the microbial degradation of nitroaromatic pollutants. The catalytic cycle of o-aminophenol dioxygenases (APDOs) has been proposed to involve formation of an Fe(II)/O2/iminobenzosemiquinone complex, although the presence of a substrate radical has been called into question by studies of related ring-cleaving dioxygenases. Recently, we reported the first synthesis of an iron(II) complex coordinated to an iminobenzosemiquinone (ISQ) ligand, namely, [Fe(Ph2Tp)(ISQtBu)] (2a; where Ph2Tp = hydrotris(3,5-diphenylpyrazol-1-yl)borate and ISQtBu is the radical anion derived from 2-amino-4,6-di-tert-butylphenol). In the current manuscript, density functional theory (DFT) calculations and a wide variety of spectroscopic methods (electronic absorption, Mössbauer, magnetic circular dichroism, and resonance Raman) were employed to obtain detailed electronic-structure descriptions of 2a and its one-electron oxidized derivative [3a]+. In addition, we describe the synthesis and characterization of a parallel series of complexes featuring the neutral supporting ligand tris(4,5-diphenyl-1-methylimidazol-2-yl)phosphine (Ph2TIP). The isomer shifts of ~0.97 mm/s obtained via Mössbauer experiments confirm that 2a (and its Ph2TIP-based analogue [2b]+) contain Fe(II) centers, and the presence of an ISQ radical was verified by analysis of the absorption spectra in light of time-dependent DFT calculations. The collective spectroscopic data indicate that one-electron oxidation of the Fe2+–ISQ complexes yields complexes ([3a]+ and [3b]2+) with electronic configurations between the Fe3+–ISQ and Fe2+–IBQ limits (IBQ = iminobenzoquinone), highlighting the ability of o-amidophenolates to access multiple oxidation states. The implications of these results for the mechanism of APDOs and other ring-cleaving dioxygenases are discussed. PMID

  11. Crystal structure of a member of a novel family of dioxygenases (PF10014) reveals a conserved cupin fold and active site

    PubMed Central

    Xu, Qingping; Grant, Joanna; Chiu, Hsiu-Ju; Farr, Carol L.; Jaroszewski, Lukasz; Knuth, Mark W.; Miller, Mitchell D.; Lesley, Scott A.; Godzik, Adam; Elsliger, Marc-André; Deacon, Ashley M.; Wilson, Ian A.

    2014-01-01

    PF10014 is a novel family of 2-oxyglutarate-Fe2+-dependent dioxygenases that are involved in biosynthesis of antibiotics and regulation of biofilm formation, likely by catalyzing hydroxylation of free amino acids or other related ligands. The crystal structure of a PF10014 member from Methylibium petroleiphilum at 1.9 Å resolution shows strong structural similarity to cupin dioxygenases in overall fold and active site, despite very remote homology. However, one of the β-strands of the cupin catalytic core is replaced by a loop that displays conformational isomerism that likely regulates the active site. PMID:23852666

  12. Crystal structure of a member of a novel family of dioxygenases (PF10014) reveals a conserved cupin fold and active site.

    PubMed

    Xu, Qingping; Grant, Joanna; Chiu, Hsiu-Ju; Farr, Carol L; Jaroszewski, Lukasz; Knuth, Mark W; Miller, Mitchell D; Lesley, Scott A; Godzik, Adam; Elsliger, Marc-André; Deacon, Ashley M; Wilson, Ian A

    2014-01-01

    PF10014 is a novel family of 2-oxyglutarate-Fe(2+) -dependent dioxygenases that are involved in biosynthesis of antibiotics and regulation of biofilm formation, likely by catalyzing hydroxylation of free amino acids or other related ligands. The crystal structure of a PF10014 member from Methylibium petroleiphilum at 1.9 Å resolution shows strong structural similarity to cupin dioxygenases in overall fold and active site, despite very remote homology. However, one of the β-strands of the cupin catalytic core is replaced by a loop that displays conformational isomerism that likely regulates the active site.

  13. Structure and mechanism leading to formation of the cysteine sulfinate product complex of a biomimetic cysteine dioxygenase model.

    PubMed

    Sallmann, Madleen; Kumar, Suresh; Chernev, Petko; Nehrkorn, Joscha; Schnegg, Alexander; Kumar, Devesh; Dau, Holger; Limberg, Christian; de Visser, Sam P

    2015-05-11

    Cysteine dioxygenase is a unique nonheme iron enzyme that is involved in the metabolism of cysteine in the body. It contains an iron active site with an unusual 3-His ligation to the protein, which contrasts with the structural features of common nonheme iron dioxygenases. Recently, some of us reported a truly biomimetic model for this enzyme, namely a trispyrazolylborato iron(II) cysteinato complex, which not only has a structure very similar to the enzyme-substrate complex but also represents a functional model: Treatment of the model with dioxygen leads to cysteine dioxygenation, as shown by isolating the cysteine part of the product in the course of the work-up. However, little is known on the conversion mechanism and, so far, not even the structure of the actual product complex had been characterised, which is also unknown in case of the enzyme. In a multidisciplinary approach including density functional theory calculations and X-ray absorption spectroscopy, we have now determined the structure of the actual sulfinato complex for the first time. The Cys-SO2 (-) functional group was found to be bound in an η(2) -O,O-coordination mode, which, based on the excellent resemblance between model and enzyme, also provides the first support for a corresponding binding mode within the enzymatic product complex. Indeed, this is again confirmed by theory, which had predicted a η(2) -O,O-binding mode for synthetic as well as the natural enzyme.

  14. Community structure and PAH ring-hydroxylating dioxygenase genes of a marine pyrene-degrading microbial consortium.

    PubMed

    Gallego, Sara; Vila, Joaquim; Tauler, Margalida; Nieto, José María; Breugelmans, Philip; Springael, Dirk; Grifoll, Magdalena

    2014-07-01

    Marine microbial consortium UBF, enriched from a beach polluted by the Prestige oil spill and highly efficient in degrading this heavy fuel, was subcultured in pyrene minimal medium. The pyrene-degrading subpopulation (UBF-Py) mineralized 31 % of pyrene without accumulation of partially oxidized intermediates indicating the cooperation of different microbial components in substrate mineralization. The microbial community composition was characterized by culture dependent and PCR based methods (PCR-DGGE and clone libraries). Molecular analyses showed a highly stable community composed by Alphaproteobacteria (84 %, Breoghania, Thalassospira, Paracoccus, and Martelella) and Actinobacteria (16 %, Gordonia). The members of Thalasosspira and Gordonia were not recovered as pure cultures, but five additional strains, not detected in the molecular analysis, that classified within the genera Novosphingobium, Sphingopyxis, Aurantimonas (Alphaproteobacteria), Alcanivorax (Gammaproteobacteria) and Micrococcus (Actinobacteria), were isolated. None of the isolates degraded pyrene or other PAHs in pure culture. PCR amplification of Gram-positive and Gram-negative dioxygenase genes did not produce results with any of the cultured strains. However, sequences related to the NidA3 pyrene dioxygenase present in mycobacterial strains were detected in UBF-Py consortium, suggesting the representative of Gordonia as the key pyrene degrader, which is consistent with a preeminent role of actinobacteria in pyrene removal in coastal environments affected by marine oil spills.

  15. Molecular basis for catalysis and substrate-mediated cellular stabilization of human tryptophan 2,3-dioxygenase

    PubMed Central

    Lewis-Ballester, Ariel; Forouhar, Farhad; Kim, Sung-Mi; Lew, Scott; Wang, YongQiang; Karkashon, Shay; Seetharaman, Jayaraman; Batabyal, Dipanwita; Chiang, Bing-Yu; Hussain, Munif; Correia, Maria Almira; Yeh, Syun-Ru; Tong, Liang

    2016-01-01

    Tryptophan 2,3-dioxygenase (TDO) and indoleamine 2,3-dioxygenase (IDO) play a central role in tryptophan metabolism and are involved in many cellular and disease processes. Here we report the crystal structure of human TDO (hTDO) in a ternary complex with the substrates L-Trp and O2 and in a binary complex with the product N-formylkynurenine (NFK), defining for the first time the binding modes of both substrates and the product of this enzyme. The structure indicates that the dioxygenation reaction is initiated by a direct attack of O2 on the C2 atom of the L-Trp indole ring. The structure also reveals an exo binding site for L-Trp, located ~42 Å from the active site and formed by residues conserved among tryptophan-auxotrophic TDOs. Biochemical and cellular studies indicate that Trp binding at this exo site does not affect enzyme catalysis but instead it retards the degradation of hTDO through the ubiquitin-dependent proteasomal pathway. This exo site may therefore provide a novel L-Trp-mediated regulation mechanism for cellular degradation of hTDO, which may have important implications in human diseases. PMID:27762317

  16. Crystallization and Preliminary X-ray Diffraction Analysis of Recombinant Chlorocatechol 1 2-dioxygenase from Pseudomonas Putida

    SciTech Connect

    J Rustiguel; M Pinheiro; A Araujo; M Nonato

    2011-12-31

    Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapor-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT - LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6{sub 1}22 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 {angstrom} resolution.

  17. Crystallization and preliminary X-ray diffraction analysis of recombinant chlorocatechol 1,2-­dioxygenase from Pseudomonas putida

    PubMed Central

    Rustiguel, Joane Kathelen; Pinheiro, Matheus Pinto; Araújo, Ana Paula Ulian; Nonato, Maria Cristina

    2011-01-01

    Chlorocatechol 1,2-dioxygenase from the Gram-negative bacterium Pseudomonas putida (Pp 1,2-CCD) is considered to be an important biotechnological tool owing to its ability to process a broad spectrum of organic pollutants. In the current work, the crystallization, crystallographic characterization and phasing of the recombinant Pp 1,2-CCD enzyme are described. Reddish-brown crystals were obtained in the presence of polyethylene glycol and magnesium acetate by utilizing the vapour-diffusion technique in sitting drops. Crystal dehydration was the key step in obtaining data sets, which were collected on the D03B-MX2 beamline at the CNPEM/MCT – LNLS using a MAR CCD detector. Pp 1,2-CCD crystals belonged to space group P6122 and the crystallographic structure of Pp 1,2-CCD has been solved by the MR-SAD technique using Fe atoms as scattering centres and the coordinates of 3-chlorocatechol 1,2-dioxygenase from Rhodococcus opacus (PDB entry 2boy) as the search model. The initial model, which contains three molecules in the asymmetric unit, has been refined to 3.4 Å resolution. PMID:21505253

  18. Multistep conversion of para-substituted phenols by phenol hydroxylase and 2,3-dihydroxybiphenyl 1,2-dioxygenase.

    PubMed

    Qu, Yuanyuan; Shi, Shengnan; Ma, Qiao; Kong, Chunlei; Zhou, Hao; Zhang, Xuwang; Zhou, Jiti

    2013-04-01

    A multistep conversion system of para-substituted phenols by recombinant phenol hydroxylase (PH(IND)) and 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC(LA-4)) was constructed in this study. Docking studies with different para-substituted phenols and corresponding catechols inside of the active site of PH(IND) and BphC(LA-4) predicted that all the substrates should be transformed. High-performance liquid chromatography-mass spectrometry analysis showed that the products of multistep conversion were the corresponding para-substituted catechols and semialdehydes. For the first-step conversion, the formation rate of 4-fluorocatechol (0.39 μM/min/mg dry weight) by strain PH(IND) hydroxylation was 1.15, 6.50, 3.00, and 1.18-fold higher than the formation of 4-chlorocatechol, 4-bromocatechol, 4-nitrocatechol, and 4-methylcatechol, respectively. For the second-step conversion, the formation rates of semialdehydes by strain BphC(LA-4) were as follows: 5-fluoro-HODA>5-chloro-HODA>2-hydroxy-5-nitro-ODA>5-bromo-HODA>2-hydroxy-5-methyl-ODA. The present study suggested that the multistep conversion by both ring hydroxylase and cleavage dioxygenase should be potential in the synthesis of industrial precursors and provide a novel avenue in the wastewater recycling treatment.

  19. Activity of a Carboxyl-Terminal Truncated Form of Catechol 2,3-Dioxygenase from Planococcus sp. S5

    PubMed Central

    2014-01-01

    Catechol 2,3-dioxygenases (C23Os, E.C.1.13.12.2) are two domain enzymes that catalyze degradation of monoaromatic hydrocarbons. The catalytically active C-domain of all known C23Os comprises ferrous ion ligands as well as residues forming active site pocket. The aim of this work was to examine and discuss the effect of nonsense mutation at position 289 on the activity of catechol 2,3-dioxygenase from Planococcus strain. Although the mutant C23O showed the same optimal temperature for activity as the wild-type protein (35°C), it exhibited activity slightly more tolerant to alkaline pH. Mutant enzyme exhibited also higher affinity to catechol as a substrate. Its Km (66.17 µM) was approximately 30% lower than that of wild-type enzyme. Interestingly, removal of the C-terminal residues resulted in 1.5- to 1.8-fold (P < 0.05) increase in the activity of C23OB61 against 4-methylcatechol and 4-chlorocatechol, respectively, while towards catechol the activity of the protein dropped to about 80% of that of the wild-type enzyme. The results obtained may facilitate the engineering of the C23O for application in the bioremediation of polluted areas. PMID:24693238

  20. Overexpression of the rice carotenoid cleavage dioxygenase 1 gene in Golden Rice endosperm suggests apocarotenoids as substrates in planta.

    PubMed

    Ilg, Andrea; Yu, Qiuju; Schaub, Patrick; Beyer, Peter; Al-Babili, Salim

    2010-08-01

    Carotenoids are converted by carotenoid cleavage dioxygenases that catalyze oxidative cleavage reactions leading to apocarotenoids. However, apocarotenoids can also be further truncated by some members of this enzyme family. The plant carotenoid cleavage dioxygenase 1 (CCD1) subfamily is known to degrade both carotenoids and apocarotenoids in vitro, leading to different volatile compounds. In this study, we investigated the impact of the rice CCD1 (OsCCD1) on the pigmentation of Golden Rice 2 (GR2), a genetically modified rice variety accumulating carotenoids in the endosperm. For this purpose, the corresponding cDNA was introduced into the rice genome under the control of an endosperm-specific promoter in sense and anti-sense orientations. Despite high expression levels of OsCCD1 in sense plants, pigment analysis revealed carotenoid levels and patterns comparable to those of GR2, pleading against carotenoids as substrates in rice endosperm. In support, similar carotenoid contents were determined in anti-sense plants. To check whether OsCCD1 overexpressed in GR2 endosperm is active, in vitro assays were performed with apocarotenoid substrates. HPLC analysis confirmed the cleavage activity of introduced OsCCD1. Our data indicate that apocarotenoids rather than carotenoids are the substrates of OsCCD1 in planta.

  1. Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

    PubMed Central

    Haigler, B E; Gibson, D T

    1990-01-01

    Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase. Images FIG. 3 PMID:2294092

  2. X-ray crystallography, mass spectrometry and single crystal microspectrophotometry: a multidisciplinary characterization of catechol 1,2 dioxygenase.

    PubMed

    Micalella, Chiara; Martignon, Sara; Bruno, Stefano; Pioselli, Barbara; Caglio, Raffaella; Valetti, Francesca; Pessione, Enrica; Giunta, Carlo; Rizzi, Menico

    2011-06-01

    Intradiol-cleaving catechol 1,2 dioxygenases are Fe(III) dependent enzymes that act on catechol and substituted catechols, including chlorocatechols pollutants, by inserting molecular oxygen in the aromatic ring. Members of this class are the object of intense biochemical investigations aimed at the understanding of their catalytic mechanism, particularly for designing mutants with selected catalytic properties. We report here an in depth investigation of catechol 1,2 dioxygenase IsoB from Acinetobacter radioresistens LMG S13 and its A72G and L69A mutants. By applying a multidisciplinary approach that includes high resolution X-rays crystallography, mass spectrometry and single crystal microspectrophotometry, we characterised the phospholipid bound to the enzyme and provided a structural framework to understand the inversion of substrate specificity showed by the mutants. Our results might be of help for the rational design of enzyme mutants showing a biotechnologically relevant substrate specificity, particularly to be used in bioremediation. This article is part of a Special Issue entitled: Protein Structure and Function in the Crystalline State.

  3. Cloning and mutagenesis of catechol 2,3-dioxygenase gene from the gram-positive Planococcus sp. strain S5.

    PubMed

    Hupert-Kocurek, Katarzyna; Stawicka, Agnieszka; Wojcieszyńska, Danuta; Guzik, Urszula

    2013-01-01

    In this study, the catechol 2,3-dioxygenase gene that encodes a 307- amino-acid protein was cloned from Planococcus sp. S5. The protein was identified to be a member of the superfamily I, subfamily 2A of extradiol dioxygenases. In order to study residues and regions affecting the enzyme's catalytic parameters, the c23o gene was randomly mutated by error-prone PCR. The wild-type enzyme and mutants containing substitutions within either the C-terminal or both domains were functionally produced in Escherichia coli and their activity towards catechol was characterized. The C23OB65 mutant with R296Q substitution showed significant tolerance to acidic pH with an optimum at pH 5.0. In addition, it showed activity more than 1.5 as high as that of the wild type enzyme and its Km was 2.5 times lower. It also showed altered sensitivity to substrate inhibition. The results indicate that residue at position 296 plays a role in determining pH dependence of the enzyme and its activity. Lower activity toward catechol was shown for mutants C23OB58 and C23OB81. Despite lower activity, these mutants showed higher affinity to catechol and were more sensitive to substrate concentration than nonmutated enzyme.

  4. Tryptophan-2,3-Dioxygenase (TDO) deficiency is associated with subclinical neuroprotection in a mouse model of multiple sclerosis

    PubMed Central

    Lanz, Tobias V.; Williams, Sarah K.; Stojic, Aleksandar; Iwantscheff, Simeon; Sonner, Jana K.; Grabitz, Carl; Becker, Simon; Böhler, Laura-Inés; Mohapatra, Soumya R.; Sahm, Felix; Küblbeck, Günter; Nakamura, Toshikazu; Funakoshi, Hiroshi; Opitz, Christiane A.; Wick, Wolfgang; Diem, Ricarda; Platten, Michael

    2017-01-01

    The catabolism of tryptophan to immunosuppressive and neuroactive kynurenines is a key metabolic pathway regulating immune responses and neurotoxicity. The rate-limiting step is controlled by indoleamine-2,3-dioxygenase (IDO) and tryptophan-2,3-dioxygenase (TDO). IDO is expressed in antigen presenting cells during immune reactions, hepatic TDO regulates blood homeostasis of tryptophan and neuronal TDO influences neurogenesis. While the role of IDO has been described in multiple immunological settings, little is known about TDO’s effects on the immune system. TDO-deficiency is neuroprotective in C. elegans and Drosophila by increasing tryptophan and specific kynurenines. Here we have determined the role of TDO in autoimmunity and neurodegeneration in experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis. We created reporter-TDO mice for in vivo imaging to show that hepatic but not CNS TDO expression is activated during EAE. TDO deficiency did not influence myelin-specific T cells, leukocyte infiltration into the CNS, demyelination and disease activity. TDO-deficiency protected from neuronal loss in the spinal cord but not in the optic nerves. While this protection did not translate to an improved overt clinical outcome, our data suggest that spatially distinct neuroprotection is conserved in mammals and support TDO as a potential target for treatment of diseases associated with neurodegeneration. PMID:28117398

  5. Functional characterisation of three members of the Vitis vinifera L. carotenoid cleavage dioxygenase gene family

    PubMed Central

    2013-01-01

    Background In plants, carotenoids serve as the precursors to C13-norisoprenoids, a group of apocarotenoid compounds with diverse biological functions. Enzymatic cleavage of carotenoids catalysed by members of the carotenoid cleavage dioxygenase (CCD) family has been shown to produce a number of industrially important volatile flavour and aroma apocarotenoids including β-ionone, geranylacetone, pseudoionone, α-ionone and 3-hydroxy-β-ionone in a range of plant species. Apocarotenoids contribute to the floral and fruity attributes of many wine cultivars and are thereby, at least partly, responsible for the “varietal character”. Despite their importance in grapes and wine; carotenoid cleavage activity has only been described for VvCCD1 and the mechanism(s) and regulation of carotenoid catabolism remains largely unknown. Results Three grapevine-derived CCD-encoding genes have been isolated and shown to be functional with unique substrate cleavage capacities. Our results demonstrate that the VvCCD4a and VvCCD4b catalyse the cleavage of both linear and cyclic carotenoid substrates. The expression of VvCCD1, VvCCD4a and VvCCD4b was detected in leaf, flower and throughout berry development. VvCCD1 expression was constitutive, whereas VvCCD4a expression was predominant in leaves and VvCCD4b in berries. A transgenic population with a 12-fold range of VvCCD1 expression exhibited a lack of correlation between VvCCD1 expression and carotenoid substrates and/or apocarotenoid products in leaves, providing proof that the in planta function(s) of VvCCD1 in photosynthetically active tissue is distinct from the in vitro activities demonstrated. The isolation and functional characterisation of VvCCD4a and VvCCD4b identify two additional CCDs that are functional in grapevine. Conclusions Taken together, our results indicate that the three CCDs are under various levels of control that include gene expression (spatial and temporal), substrate specificity and compartmentalisation

  6. NO Binding to Mn-Substituted Homoprotocatechuate 2,3-Dioxygenase: Relationship to O2 Reactivity

    PubMed Central

    Hayden, Joshua A.; Farquhar, Erik R.; Que, Lawrence; Lipscomb, John D.; Hendrich, Michael P.

    2014-01-01

    Homoprotocatechuate 2,3-dioxygenase (FeHPCD) activates O2 to catalyze the aromatic ring opening of 3,4-dihydroxyphenylacetic acid (HPCA). The enzyme requires FeII for catalysis, but MnII can be substituted (MnHPCD) with essentially no change in the steady-state kinetic parameters. Near simultaneous O2 and HPCA activation has been proposed to occur through transfer of an electron(s) from HPCA to O2 through the divalent metal. In O2 reactions with MnHPCD-HPCA and the 4-nitrocatechol (4NC) complex of the His200Asn (H200N) variant of FeHPCD, this transfer has resulted in the detection of a transient MIII-O2•− species not observed during turnover of the wild type FeHPCD. The factors governing formation of the MIII-O2•− species are explored here with EPR spectroscopy using MnHPCD and nitric oxide (NO) as an O2 surrogate. Both the HPCA and dihydroxymandelic substrate complexes of MnHPCD bind NO, thus representing the first reported stable MnNO complexes of a nonheme enzyme. In contrast, the free enzyme, the MnHPCD-4NC complex, and the MnH200N and MnH200Q variants with or without HPCA bound do not bind NO. The MnHPCD-ligand complexes that bind NO are also active in normal O2-linked turnover, whereas the others are inactive. Past studies have shown that FeHPCD and the analogous variants and catecholic ligand complexes all bind NO, and are active in normal turnover. This contrasting behavior may stem from ability of the enzyme to maintain the ~0.8 V difference in the solution redox potentials of FeII and MnII. Due to the higher potential of Mn, the formation of the NO or O2 adduct requires both strong charge donation from the bound catecholic ligand and additional stabilization by interaction with the active site His200. The same non-optimal electronic and structural forces that prevent NO and O2 binding in MnHPCD variants may lead to inefficient electron transfer from the catecholic substrate to the metal center in variants of FeHPCD during O2-linked turnover

  7. Benzene-Induced Uncoupling of Naphthalene Dioxygenase Activity and Enzyme Inactivation by Production of Hydrogen Peroxide

    PubMed Central

    Lee, Kyoung

    1999-01-01

    Naphthalene dioxygenase (NDO) is a multicomponent enzyme system that oxidizes naphthalene to (+)-cis-(1R,2S)-1,2-dihydroxy-1,2-dihydronaphthalene with consumption of O2 and two electrons from NAD(P)H. In the presence of benzene, NADH oxidation and O2 utilization were partially uncoupled from substrate oxidation. Approximately 40 to 50% of the consumed O2 was detected as hydrogen peroxide. The rate of benzene-dependent O2 consumption decreased with time, but it was partially increased by the addition of catalase in the course of the O2 consumption by NDO. Detailed experiments showed that the total amount of O2 consumed and the rate of benzene-induced O2 consumption increased in the presence of hydrogen peroxide-scavenging agents, and further addition of the terminal oxygenase component (ISPNAP) of NDO. Kinetic studies showed that ISPNAP was irreversibly inactivated in the reaction that contained benzene, but the inactivation was relieved to a high degree in the presence of catalase and partially relieved in the presence of 0.1 mM ferrous ion. Benzene- and naphthalene-reacted ISPNAP gave almost identical visible absorption spectra. In addition, hydrogen peroxide added at a range of 0.1 to 0.6 mM to the reaction mixtures inactivated the reduced ISPNAP containing mononuclear iron. These results show that hydrogen peroxide released during the uncoupling reaction acts both as an inhibitor of benzene-dependent O2 consumption and as an inactivator of ISPNAP. It is proposed that the irreversible inactivation of ISPNAP occurs by a Fenton-type reaction which forms a strong oxidizing agent, hydroxyl radicals (·OH), from the reaction of hydrogen peroxide with ferrous mononuclear iron at the active site. Furthermore, when [14C]benzene was used as the substrate, cis-benzene 1,2-dihydrodiol formed by NDO was detected. This result shows that NDO also couples a trace amount of benzene to both O2 consumption and NADH oxidation. PMID:10217759

  8. Novel aromatic ring-hydroxylating dioxygenase genes from coastal marine sediments of Patagonia

    PubMed Central

    Lozada, Mariana; Riva Mercadal, Juan P; Guerrero, Leandro D; Di Marzio, Walter D; Ferrero, Marcela A; Dionisi, Hebe M

    2008-01-01

    Background Polycyclic aromatic hydrocarbons (PAHs), widespread pollutants in the marine environment, can produce adverse effects in marine organisms and can be transferred to humans through seafood. Our knowledge of PAH-degrading bacterial populations in the marine environment is still very limited, and mainly originates from studies of cultured bacteria. In this work, genes coding catabolic enzymes from PAH-biodegradation pathways were characterized in coastal sediments of Patagonia with different levels of PAH contamination. Results Genes encoding for the catalytic alpha subunit of aromatic ring-hydroxylating dioxygenases (ARHDs) were amplified from intertidal sediment samples using two different primer sets. Products were cloned and screened by restriction fragment length polymorphism analysis. Clones representing each restriction pattern were selected in each library for sequencing. A total of 500 clones were screened in 9 gene libraries, and 193 clones were sequenced. Libraries contained one to five different ARHD gene types, and this number was correlated with the number of PAHs found in the samples above the quantification limit (r = 0.834, p < 0.05). Overall, eight different ARHD gene types were detected in the sediments. In five of them, their deduced amino acid sequences formed deeply rooted branches with previously described ARHD peptide sequences, exhibiting less than 70% identity to them. They contain consensus sequences of the Rieske type [2Fe-2S] cluster binding site, suggesting that these gene fragments encode for ARHDs. On the other hand, three gene types were closely related to previously described ARHDs: archetypical nahAc-like genes, phnAc-like genes as identified in Alcaligenes faecalis AFK2, and phnA1-like genes from marine PAH-degraders from the genus Cycloclasticus. Conclusion These results show the presence of hitherto unidentified ARHD genes in this sub-Antarctic marine environment exposed to anthropogenic contamination. This information

  9. Expression and Prognostic Value of Indoleamine 2,3-dioxygenase in Pancreatic Cancer

    PubMed Central

    Zhang, Tao; Tan, Xiang-Long; Xu, Yong; Wang, Zi-Zheng; Xiao, Chao-Hui; Liu, Rong

    2017-01-01

    Background: Indoleamine 2,3-dioxygenase (IDO), an enzyme for tryptophan metabolism through the kynurenine pathway, exhibits an immunosuppressive effect and induces immune tolerance in tumor cells. The effects of IDO on pancreatic cancer are poorly understood. This study aimed to investigate the expression and prognostic significance of IDO in pancreatic cancer. Methods: We evaluated the protein expression of IDO in PANC-1, CFPAC-1, and BxPC-3 cell lines with or without 48 h treatment by 500 U/ml interferon-γ (IFN-γ). We performed immunohistochemical staining and Western blot analysis for IDO expression in both pancreatic cancer and normal pancreas tissues obtained from Chinese PLA General Hospital from July 2012 to December 2013. Survival analysis was performed to correlate IDO expression and histopathologic parameters with overall survival. The Kaplan-Meier method and Cox proportional hazards regression model were conducted. Results: PANC-1, CFPAC-1, and BxPC-3 cell lines expressed IDO at the protein level, and the relative expression amount increased after stimulation with 500 U/ml IFN-γ. Immunohistochemical analysis results revealed that high IDO expression was observed in 59% of pancreatic adenocarcinoma tissues. Compared with normal pancreatic tissues, pancreatic adenocarcinoma showed significantly higher IDO expression levels, especially among patients with high tumor node metastasis (TNM) stages (χ2 = 4.550, P = 0.030), poor histological differentiation (χ2 = 5.690, P = 0.017), and lymph node metastasis (χ2 = 4.340 P = 0.037). Kaplan-Meier survival curves showed that high IDO expression was correlated with low survival rates (hazard ratio [HR] = 0.49 P = 0.009). Multivariate analysis using Cox proportional hazards model indicated that lymph node metastasis (HR = 0.35 P = 0.010) and IDO expression (HR = 0.42 P = 0.020) were two independent prognostic predictors of pancreatic adenocarcinoma. Conclusions: The study confirmed that high IDO expression in

  10. NUCLEOTIDE SEQUENCING AND TRANSCRIPTIONAL MAPPING OF THE GENES ENCODING BIPHENYL DIOXYGENASE, A MULTICOM- PONENT POLYCHLORINATED-BIPHENYL-DEGRADING ENZYME IN PSEUDOMONAS STRAIN LB400

    EPA Science Inventory

    The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxy...

  11. Constitutive Expression of a Nag-Like Dioxygenase Gene through an Internal Promoter in the 2-Chloronitrobenzene Catabolism Gene Cluster of Pseudomonas stutzeri ZWLR2-1

    PubMed Central

    Gao, Yi-Zhou; Liu, Hong; Chao, Hong-Jun

    2016-01-01

    ABSTRACT The gene cluster encoding the 2-chloronitrobenzene (2CNB) catabolism pathway in Pseudomonas stutzeri ZWLR2-1 is a patchwork assembly of a Nag-like dioxygenase (dioxygenase belonging to the naphthalene dioxygenase NagAaAbAcAd family from Ralstonia sp. strain U2) gene cluster and a chlorocatechol catabolism cluster. However, the transcriptional regulator gene usually present in the Nag-like dioxygenase gene cluster is missing, leaving it unclear how this cluster is expressed. The pattern of expression of the 2CNB catabolism cluster was investigated here. The results demonstrate that the expression was constitutive and not induced by its substrate 2CNB or salicylate, the usual inducer of expression in the Nag-like dioxygenase family. Reverse transcription-PCR indicated the presence of at least one transcript containing all the structural genes for 2CNB degradation. Among the three promoters verified in the gene cluster, P1 served as the promoter for the entire catabolism operon, but the internal promoters P2 and P3 also enhanced the transcription of the genes downstream. The P3 promoter, which was not previously defined as a promoter sequence, was the strongest of these three promoters. It drove the expression of cnbAcAd encoding the dioxygenase that catalyzes the initial reaction in the 2CNB catabolism pathway. Bioinformatics and mutation analyses suggested that this P3 promoter evolved through the duplication of an 18-bp fragment and introduction of an extra 132-bp fragment. IMPORTANCE The release of many synthetic compounds into the environment places selective pressure on bacteria to develop their ability to utilize these chemicals to grow. One of the problems that a bacterium must surmount is to evolve a regulatory device for expression of the corresponding catabolism genes. Considering that 2CNB is a xenobiotic that has existed only since the onset of synthetic chemistry, it may be a good example for studying the molecular mechanisms underlying rapid

  12. Molecular cloning and expression of genes encoding a novel dioxygenase involved in low- and high-molecular-weight polycyclic aromatic hydrocarbon degradation in Mycobacterium vanbaalenii PYR-1.

    PubMed

    Kim, Seong-Jae; Kweon, Ohgew; Freeman, James P; Jones, Richard C; Adjei, Michael D; Jhoo, Jin-Woo; Edmondson, Ricky D; Cerniglia, Carl E

    2006-02-01

    Mycobacterium vanbaalenii PYR-1 is able to metabolize a wide range of low- and high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A 20-kDa protein was upregulated in PAH-metabolizing M. vanbaalenii PYR-1 cells compared to control cultures. The differentially expressed protein was identified as a beta subunit of the terminal dioxygenase using mass spectrometry. PCR with degenerate primers designed based on de novo sequenced peptides and a series of plaque hybridizations were done to screen the M. vanbaalenii PYR-1 genomic library. The genes, designated nidA3B3, encoding the alpha and beta subunits of terminal dioxygenase, were subsequently cloned and sequenced. The deduced enzyme revealed close similarities to the corresponding PAH ring-hydroxylating dioxygenases from Mycobacterium and Rhodococcus spp. but had the highest similarity, 61.9%, to the alpha subunit from Nocardioides sp. strain KP7. The alpha subunit also showed 52% sequence homology with the previously reported NidA from M. vanbaalenii PYR-1. The genes nidA3B3 were subcloned into the expression vector pET-17b, and the enzyme activity in Escherichia coli cells was reconstituted through coexpression with the ferredoxin (PhdC) and ferredoxin reductase (PhdD) genes of the phenanthrene dioxygenase from Nocardioides sp. strain KP7. The recombinant PAH dioxygenase appeared to favor the HMW PAH substrates fluoranthene, pyrene, and phenanthrene. Several other PAHs, including naphthalene, anthracene, and benz[a]anthracene, were also converted to their corresponding cis-dihydrodiols. The recombinant E. coli, however, did not show any dioxygenation activity for phthalate and biphenyl. The upregulation of nidA3B3 in M. vanbaalenii PYR-1 induced by PAHs was confirmed by reverse transcription-PCR analysis.

  13. Purification and characterization of catechol 1,2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 and cloning and sequencing of its catA gene.

    PubMed Central

    Strachan, P D; Freer, A A; Fewson, C A

    1998-01-01

    A method was developed for the purification of catechol 1, 2-dioxygenase from Rhodococcus rhodochrous NCIMB 13259 that had been grown in the presence of benzyl alcohol. The enzyme has very similar apparent Km (1-2 microM) and Vmax (13-19 units/mg of protein) values for the intradiol cleavage of catechol, 3-methylcatechol and 4-methylcatechol and it is optimally active at pH9. Cross-linking studies indicate that the enzyme is a homodimer. It contains 0.6 atoms of Fe per subunit. The enzyme was crystallized with 15% (w/v) poly(ethylene glycol) 4000/0.33 M CaCl2/25 mM Tris (pH7.5) by using a microseeding technique. Preliminary X-ray characterization showed that the crystals are in space group C2 with unit-cell dimensions a=111.9 A, b=78.1 A, c=134.6 A, beta=100 degrees. An oligonucleotide probe, made by hemi-nested PCR, was used to clone the gene encoding catechol 1,2-dioxygenase (catA). The deduced 282-residue sequence corresponds to a protein of molecular mass 31539 Da, close to the molecular mass of 31558 Da obtained by electrospray MS of the purified enzyme. catA was subcloned into the expression vector pTB361, allowing the production of catechol 1,2-dioxygenase to approx. 40% of the total cellular protein. The deduced amino acid sequence of the enzyme has 56% and 75% identity with the catechol 1, 2-dioxygenases of Arthrobacter mA3 and Rhodococcus erythropolis AN-13 respectively, but less than 35% identity with intradiol catechol and chlorocatechol dioxygenases of Gram-negative bacteria. PMID:9677336

  14. Pseudomonas aeruginosa 142 uses a three-component ortho-halobenzoate 1,2-dioxygenase for metabolism of 2,4-dichloro- and 2-chlorobenzoate.

    PubMed Central

    Romanov, V; Hausinger, R P

    1994-01-01

    Cell extracts of Pseudomonas aeruginosa 142, which was previously isolated from a polychlorinated biphenyl-degrading consortium, were shown to degrade 2,4-dichlorobenzoate, 2-chlorobenzoate, and a variety of other substituted ortho-halobenzoates by a reaction that requires oxygen, NADH, Fe(II), and flavin adenine dinucleotide. By using extracts that were chromatographically depleted of chlorocatechol and catechol 1,2-dioxygenase activities, products of the initial reaction with 2,4- or 2,5-dichlorobenzoate and 2-chlorobenzoate were identified by mass spectrometry as 4-chlorocatechol and catechol. In contrast to the well-characterized benzoate dioxygenases or the recently described 2-halobenzoate 1,2-dioxygenase from P. cepacia 2CBS (S. Fetzner, R. Müller, and F. Lingens, J. Bacteriol. 174:279-290, 1992) that possess two protein components, the P. aeruginosa enzyme was resolved by ion-exchange chromatography into three components, each of which is required for activity. To verify the distinct nature of this enzyme, we purified, characterized, and identified one component as a ferredoxin (M(r), approximately 13,000) containing a single [2Fe-2S] Rieske-type cluster (electron paramagnetic resonance spectroscopic values of gx = 1.82, gy = 1.905, and gz = 2.02 in the reduced state) that is related in sequence to ferredoxins found in the naphthalene and biphenyl three-component dioxygenase systems. By analogy to these enzymes, we propose that the P. aeruginosa ferredoxin serves as an electron carrier between an NADH-dependent ferredoxin reductase and the terminal component of the ortho-halobenzoate 1,2-dioxygenase. The broad specificity and high regiospecificity of the enzyme make it a promising candidate for use in the degradation of mixtures of chlorobenzoates. PMID:8195093

  15. Ascorbate as a co-factor for fe- and 2-oxoglutarate dependent dioxygenases: physiological activity in tumor growth and progression.

    PubMed

    Kuiper, Caroline; Vissers, Margreet C M

    2014-01-01

    Ascorbate is a specific co-factor for a large family of enzymes known as the Fe- and 2-oxoglutarate-dependent dioxygenases. These enzymes are found throughout biology and catalyze the addition of a hydroxyl group to various substrates. The proline hydroxylase that is involved in collagen maturation is well known, but in recent times many new enzymes and functions have been uncovered, including those involved in epigenetic control and hypoxia-inducible factor (HIF) regulation. These discoveries have provided crucial mechanistic insights into how ascorbate may affect tumor biology. In particular, there is growing evidence that HIF-1-dependent tumor progression may be inhibited by increasing tumor ascorbate levels. However, rigorous clinical intervention studies are lacking. This review will explore the physiological role of ascorbate as an enzyme co-factor and how this mechanism relates to cancer biology and treatment. The use of ascorbate in cancer should be informed by clinical studies based on such mechanistic hypotheses.

  16. Papaverine 7-O-demethylase, a novel 2-oxoglutarate/Fe(2+)-dependent dioxygenase from opium poppy.

    PubMed

    Farrow, Scott C; Facchini, Peter J

    2015-09-14

    Opium poppy (Papaver somniferum) produces several pharmacologically important benzylisoquinoline alkaloids including the vasodilator papaverine. Pacodine and palaudine are tri-O-methylated analogs of papaverine, which contains four O-linked methyl groups. However, the biosynthetic origin of pacodine and palaudine has not been established. Three members of the 2-oxoglutarate/Fe(2+)-dependent dioxygenases (2ODDs) family in opium poppy display widespread O-dealkylation activity on several benzylisoquinoline alkaloids with diverse structural scaffolds, and two are responsible for the antepenultimate and ultimate steps in morphine biosynthesis. We report a novel 2ODD from opium poppy catalyzing the efficient substrate- and regio-specific 7-O-demethylation of papaverine yielding pacodine. The occurrence of papaverine 7-O-demethylase (P7ODM) expands the enzymatic scope of the 2ODD family in opium poppy and suggests an unexpected biosynthetic route to pacodine.

  17. Inflammation-induced activation of the indoleamine 2,3-dioxygenase pathway: Relevance to cancer-related fatigue.

    PubMed

    Kim, Sangmi; Miller, Brian J; Stefanek, Michael E; Miller, Andrew H

    2015-07-01

    Cancer-related fatigue (CRF) is a common complication of cancer and its treatment that can significantly impair quality of life. Although the specific mechanisms remain poorly understood, inflammation is now considered to be a distinct component of CRF in addition to effects of depression, anxiety, insomnia, and other factors. One key biological pathway that may link inflammation and CRF is indoleamine 2,3-dioxygenase (IDO). Induced by inflammatory stimuli, IDO catabolizes tryptophan to kynurenine (KYN), which is subsequently converted into neuroactive metabolites. Here we summarize current knowledge concerning the relevance of the IDO pathway to CRF, including activation of the IDO pathway in cancer patients and, as a consequence, accumulation of neurotoxic KYN metabolites and depletion of serotonin in the brain. Because IDO inhibitors are already being evaluated as therapeutic agents in cancer, the elucidation of the relationship between IDO activation and CRF in cancer patients may lead to novel diagnostic and clinical approaches to managing CRF and its debilitating consequences.

  18. The Deficiency of Indoleamine 2,3-Dioxygenase Aggravates the CCl4-Induced Liver Fibrosis in Mice

    PubMed Central

    Ogiso, Hideyuki; Ito, Hiroyasu; Ando, Tatsuya; Arioka, Yuko; Kanbe, Ayumu; Ando, Kazuki; Ishikawa, Tetsuya; Saito, Kuniaki; Hara, Akira; Moriwaki, Hisataka; Shimizu, Masahito; Seishima, Mitsuru

    2016-01-01

    In the present study, we examined the role of indoleamine 2,3-dioxygenase (IDO) in the development of CCl4-induced hepatic fibrosis. The liver fibrosis induced by repetitive administration with CCl4 was aggravated in IDO-KO mice compared to WT mice. In IDO-KO mice treated with CCl4, the number of several inflammatory cells and the expression of pro-inflammatory cytokines increased in the liver. In the results, activated hepatic stellate cells (HSCs) and fibrogenic factors on HSCs increased after repetitive CCl4 administration in IDO-KO mice compared to WT mice. Moreover, the treatment with l-tryptophan aggravated the CCl4-induced hepatic fibrosis in WT mice. Our findings demonstrated that the IDO deficiency enhanced the inflammation in the liver and aggravated liver fibrosis in repetitive CCl4-treated mice. PMID:27598994

  19. [Indoleamine 2,3-Dioxygenase Activity during Fulvestrant Therapy for Aromatase Inhibitor-Resistant Metastatic Breast Cancer].

    PubMed

    Sakurai, Kenichi; Fujisaki, Shigeru; Suzuki, Shuhei; Adachi, Keita; Nagashima, Saki; Masuo, Yuki; Tomita, Ryouichi; Gonda, Kenji; Enomoto, Katsuhisa; Amano, Sadao; Matsuo, Sadanori; Umeda, Nao

    2015-10-01

    We evaluated the clinical significance of indoleamine 2,3-dioxygenase (IDO) during fulvestrant therapy for aromatase inhibitor (AI)-resistant metastatic breast cancer. IDO activity can be measured by the tryptophan (Trp)/kynurenine (Kyn) ratio. Trp and Kyn were measured with high performance liquid chromatography (HPLC). Patients with AI resistant metastatic breast cancer had a 28.6% response rate to fulvestrant therapy, and the clinical benefit rate was 76.2%. AI-resistant metastatic breast cancer patients with distant metastases had a lower serum Trp/Kyn level than patients who had local recurrences. During fulvestrant therapy, IDO activity significantly decreased in the fulvestrant responder group compared to that in the fulvestrant non-responder group. During fulvestrant therapy, the IDO activity correlated with the number of metastatic lesions. These results suggest that measuring the Trp/Kyn ratio is useful for evaluating immunological metastatic status during endocrine therapy.

  20. Discovery and characterisation of hydrazines as inhibitors of the immune suppressive enzyme, indoleamine 2,3-dioxygenase 1 (IDO1).

    PubMed

    Fung, Sai-Parng S; Wang, Haiyan; Tomek, Petr; Squire, Christopher J; Flanagan, Jack U; Palmer, Brian D; Bridewell, David J A; Tijono, Sofian M; Jamie, Joanne F; Ching, Lai-Ming

    2013-12-15

    Screening of a fragment library identified 2-hydrazinobenzothiazole as a potent inhibitor of indoleamine 2,3-dioxygenase 1 (IDO1), an enzyme expressed by tumours that suppresses the immune system. Spectroscopic studies indicated that 2-hydrazinobenzothiazole interacted with the IDO1 haem and in silico docking predicted that the interaction was through hydrazine. Subsequent studies of hydrazine derivatives identified phenylhydrazine (IC50=0.25 ± 0.07 μM) to be 32-fold more potent than 2-hydrazinobenzothiazole (IC50=8.0 ± 2.3 μM) in inhibiting rhIDO1 and that it inhibited cellular IDO1 at concentrations that were noncytotoxic to cells. Here, phenylhydrazine is shown to inhibit IDO1 through binding to haem.

  1. The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond.

    PubMed

    Fedeles, Bogdan I; Singh, Vipender; Delaney, James C; Li, Deyu; Essigmann, John M

    2015-08-21

    The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli "adaptive response" protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1-8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins.

  2. Identification of amino acid residues essential for catalytic activity of gentisate 1,2-dioxygenase from Pseudomonas alcaligenes NCIB 9867.

    PubMed

    Chua, C H; Feng, Y; Yeo, C C; Khoo, H E; Poh, C L

    2001-10-16

    Gentisate 1,2-dioxygenase (GDO, EC 1.13.11.4) is a ring cleavage enzyme that utilizes gentisate as a substrate yielding maleylpyruvate as the ring fission product. Mutant GDOs were generated by both random mutagenesis and site-directed mutagenesis of the gene cloned from Pseudomonas alcaligenes NCIB 9867. Alignment of known GDO sequences indicated the presence of a conserved central core region. Mutations generated within this central core resulted in the complete loss of enzyme activity whereas mutations in the flanking regions yielded GDOs with enzyme activities that were reduced by up to 78%. Site-directed mutagenesis was also performed on a pair of highly conserved HRH and HXH motifs found within this core region. Conversion of these His residues to Asp resulted in the complete loss of catalytic activity. Mutagenesis within the core region could have affected quaternary structure formation as well as cofactor binding. A mutant enzyme with increased catalytic activities was also characterized.

  3. The AlkB Family of Fe(II)/α-Ketoglutarate-dependent Dioxygenases: Repairing Nucleic Acid Alkylation Damage and Beyond*

    PubMed Central

    Fedeles, Bogdan I.; Singh, Vipender; Delaney, James C.; Li, Deyu; Essigmann, John M.

    2015-01-01

    The AlkB family of Fe(II)- and α-ketoglutarate-dependent dioxygenases is a class of ubiquitous direct reversal DNA repair enzymes that remove alkyl adducts from nucleobases by oxidative dealkylation. The prototypical and homonymous family member is an Escherichia coli “adaptive response” protein that protects the bacterial genome against alkylation damage. AlkB has a wide variety of substrates, including monoalkyl and exocyclic bridged adducts. Nine mammalian AlkB homologs exist (ALKBH1–8, FTO), but only a subset functions as DNA/RNA repair enzymes. This minireview presents an overview of the AlkB proteins including recent data on homologs, structural features, substrate specificities, and experimental strategies for studying DNA repair by AlkB family proteins. PMID:26152727

  4. In Vivo Self-Hydroxylation of an Iron-Substituted Manganese-Dependent Extradiol Cleaving Catechol Dioxygenase

    PubMed Central

    Farquhar, Erik R.; Emerson, Joseph P.; Koehntop, Kevin D.; Reynolds, Mark F.; Trmčić, Milena

    2011-01-01

    The homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformis (MndD) catalyzes the oxidative ring cleavage reaction of its catechol substrate in an extradiol fashion. While this reactivity is more typically associated with nonheme iron enzymes, MndD exhibits an unusual specificity for manganese(II). MndD is structurally very similar to the iron(II)-dependent homoprotocatechuate 2,3-dioxygenase from Brevibacterium fuscum (HPCD), and we have previously shown that both MndD and HPCD are equally active towards substrate turnover with either Fe(II) or Mn(II) [J P Emerson, et al. (2008) Proc Natl Acad Sci USA, 105: 7347–7352]. However, expression of MndD in E. coli under aerobic conditions in the presence of excess iron results in the isolation of inactive blue-green Fe-substituted MndD (BG-FeMndD). Spectroscopic studies indicate that this form of Fe-substituted MndD contains an Fe(III) center with a bound catecholate, which is presumably generated by in vivo self-hydroxylation of a second-sphere tyrosine residue, as found for other self-hydroxylated nonheme iron oxygenases,. The absence of this modification in either the native Mn-containing MndD or Fe-containing HPCD suggests that the metal center of Fe-substituted MndD is able to bind and activate O2 in the absence of its substrate, employing a high-valent oxoiron oxidant to carry out the observed self-hydroxylation chemistry. These results demonstrate that the active site metal in MndD can support two dramatically different O2 activation pathways, further highlighting the catalytic flexibility of enzymes containing a 2-His-1-carboxylate facial triad metal binding motif. PMID:21279661

  5. Rate-determining Attack on Substrate Precedes Rieske Cluster Oxidation during cis-Dihydroxylation by Benzoate Dioxygenase

    PubMed Central

    Rivard, Brent S.; Rogers, Melanie S.; Marell, Daniel J.; Neibergall, Matthew B.; Chakrabarty, Sarmistha; Cramer, Christopher J.; Lipscomb, John D.

    2015-01-01

    Rieske dearomatizing dioxygenases utilize a Rieske iron-sulfur cluster and a mononuclear Fe(II) located 15 Å across a subunit boundary to catalyze O2-dependent formation of cis-dihydrodiol products from aromatic substrates. During catalysis, O2 binds to the Fe(II) while the substrate bind nearby. Single turnover reactions have shown that one electron from each metal center is required for catalysis. This finding suggested that the reactive intermediate is Fe(III)-(H)peroxo or HO-Fe(V)=O formed by O-O bond scission. Surprisingly, several kinetic phases were observed during the single turnover Rieske cluster oxidation. Here, the Rieske cluster oxidation and product formation steps of a single turnover of benzoate 1,2-dioxygenase are investigated using benzoate and three fluorinated analogs. It is shown that the rate constant for product formation correlates with the reciprocal relaxation time of only the fastest kinetic phase (RRT-1) for each substrate, suggesting that the slower phases are not mechanistically relevant. RRT-1 is strongly dependent on substrate type, suggesting a role for substrate in electron transfer from the Rieske cluster to the mononuclear iron site. This insight, together with the substrate and O2 concentration dependencies of RRT-1, indicates that a reactive species is formed after substrate and O2 binding, but before electron transfer from the Rieske cluster. Computational studies show that RRT-1 is correlated with the electron density at the substrate carbon closest to the Fe(II), consistent with initial electrophilic attack by an Fe(III)-superoxo intermediate. The resulting Fe(III)-peroxo-aryl radical species would then readily accept an electron from the Rieske cluster to complete the cis-dihydroxylation reaction. PMID:26154836

  6. Characterization of the fungal gibberellin desaturase as a 2-oxoglutarate-dependent dioxygenase and its utilization for enhancing plant growth.

    PubMed

    Bhattacharya, Anjanabha; Kourmpetli, Sofia; Ward, Dennis A; Thomas, Stephen G; Gong, Fan; Powers, Stephen J; Carrera, Esther; Taylor, Benjamin; de Caceres Gonzalez, Francisco Nuñez; Tudzynski, Bettina; Phillips, Andrew L; Davey, Michael R; Hedden, Peter

    2012-10-01

    The biosynthesis of gibberellic acid (GA(3)) by the fungus Fusarium fujikuroi is catalyzed by seven enzymes encoded in a gene cluster. While four of these enzymes are characterized as cytochrome P450 monooxygenases, the nature of a fifth oxidase, GA(4) desaturase (DES), is unknown. DES converts GA(4) to GA(7) by the formation of a carbon-1,2 double bond in the penultimate step of the pathway. Here, we show by expression of the des complementary DNA in Escherichia coli that DES has the characteristics of a 2-oxoglutarate-dependent dioxygenase. Although it has low amino acid sequence homology with known 2-oxoglutarate-dependent dioxygenases, putative iron- and 2-oxoglutarate-binding residues, typical of such enzymes, are apparent in its primary sequence. A survey of sequence databases revealed that homologs of DES are widespread in the ascomycetes, although in most cases the homologs must participate in non-gibberellin (GA) pathways. Expression of des from the cauliflower mosaic virus 35S promoter in the plant species Solanum nigrum, Solanum dulcamara, and Nicotiana sylvestris resulted in substantial growth stimulation, with a 3-fold increase in height in S. dulcamara compared with controls. In S. nigrum, the height increase was accompanied by a 20-fold higher concentration of GA(3) in the growing shoots than in controls, although GA(1) content was reduced. Expression of des was also shown to partially restore growth in plants dwarfed by ectopic expression of a GA 2-oxidase (GA-deactivating) gene, consistent with GA(3) being protected from 2-oxidation. Thus, des has the potential to enable substantial growth increases, with practical implications, for example, in biomass production.

  7. Regioselectivity and Enantioselectivity of Naphthalene Dioxygenase during Arene cis-Dihydroxylation: Control by Phenylalanine 352 in the α Subunit

    PubMed Central

    Parales, Rebecca E.; Resnick, Sol M.; Yu, Chi-Li; Boyd, Derek R.; Sharma, Narain D.; Gibson, David T.

    2000-01-01

    The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the α subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl cis-3,4-dihydrodiol [77 and 60% (−)-(3S,4R), respectively] to that formed by wild-type NDO [>98% (+)-(3R,4S)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene cis-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoikuyae B8/36. A recombinant Escherichia coli strain expressing the F352V variant of NDO and the enantioselective toluene cis-dihydrodiol dehydrogenase from Pseudomonas putida F1 was used to produce enantiomerically pure (−)-biphenyl cis-(3S,4R)-dihydrodiol and (−)-phenanthrene cis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively. PMID:10986254

  8. Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes.

    PubMed

    Ledesma-García, Laura; Sánchez-Azqueta, Ana; Medina, Milagros; Reyes-Ramírez, Francisca; Santero, Eduardo

    2016-03-31

    Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H-ThnA4-ThnA3-ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H-ThnA4-ThnA3-ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction.

  9. Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400.

    PubMed Central

    Haddock, J D; Gibson, D T

    1995-01-01

    The iron-sulfur protein of biphenyl 2,3-dioxygenase (ISPBPH) was purified from Pseudomonas sp. strain LB400. The protein is composed of a 1:1 ratio of a large (alpha) subunit with an estimated molecular weight of 53,300 and a small (beta) subunit with an estimated molecular weight of 27,300. The native molecular weight was 209,000, indicating that the protein adopts an alpha 3 beta 3 native conformation. Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of alpha beta heterodimer. The absorbance spectrum showed peaks at 325 and 450 nm with a broad shoulder at 550 nm. The spectrum was bleached upon reduction of the protein with NADPH in the presence of catalytic amounts of ferredoxinBPH and ferredoxinBPH oxidoreductase. The electron paramagnetic resonance spectrum of the reduced protein showed three signals at gx = 1.74, gy = 1.92, and gz = 2.01. These properties are characteristic of proteins that contain a Rieske-type [2Fe-2S] center. Biphenyl was oxidized to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene by ISPBPH in the presence of ferredoxinBPH, ferredoxinBPH oxidoreductase, NADPH, and ferrous iron. Naphthalene was also oxidized to a cis-dihydrodiol, but only 3% was converted to product under the same conditions that gave 92% oxidation of biphenyl. Benzene, toluene, 2,5-dichlorotoluene, carbazole, and dibenzothiophene were not oxidized. ISPBPH is proposed to be the terminal oxygenase component of biphenyl 2,3-dioxygenase where substrate binding and oxidation occur via addition of molecular oxygen and two reducing equivalents. PMID:7592331

  10. Localization of the human indoleamine 2,3-dioxygenase (IDO) gene to the pericentromeric region of human chromosome 8

    SciTech Connect

    Burkin, D.J.; Jones, C. ); Kimbro, K.S.; Taylor, M.W. ); Barr, B.L.; Gupta, S.L. )

    1993-07-01

    Indoleamine 2,3-dioxygenase (IDO) is the first enzyme in the catabolic pathway for tryptophan. This extrahepatic enzyme differs from the hepatic enzyme, tryptophan 2,3-dioxygenase (TDO), in molecular as well as enzymatic characteristics, although both enzymes catalyze the same reaction: cleavage of tryptophan into N-formylkynurenine. The induction of IDO by IFN-[gamma] plays a role in the antigrowth effect of IFN-[gamma] in cell cultures and in the inhibition of intracellular pathogens, e.g., Toxoplasma gondii and Chlamydia psittaci. Tryptophan is also the precursor for the synthesis of serotonin, and reduced levels of tryptophan and serotonin found in AIDS patients have been correlated with the presence of IFN-[gamma] and consequent elevation of IDO activity. The IDO enzyme has been purified and characterized, and its cDNA and genomic DNA clones have been isolated and analyzed. DNA from hybrid cells containing fragments of human chromosome 8 was used to determine the regional localization of the IDO gene on chromosome 8. The hybrids R30-5B and R30-2A contain 8p11 [yields] qter and 8q13 [yields] qter, respectively. Hybrid 229-3A contains the 8pter [yields] q11. The hybrid R30-2A was negative for the IDO gene, whereas R30-5B and 229-3A were positive as analyzed by PCR and verified by Southern blotting. Only the region close to the centromere is shared by R30-5B and 229-3A hybrids. The results indicate that the IDO gene is located on chromosome 8p11 [yields] q11.

  11. Cytosolic and plastoglobule-targeted carotenoid dioxygenases from Crocus sativus are both involved in beta-ionone release.

    PubMed

    Rubio, Angela; Rambla, José Luís; Santaella, Marcella; Gómez, M Dolores; Orzaez, Diego; Granell, Antonio; Gómez-Gómez, Lourdes

    2008-09-05

    Saffron, the processed stigma of Crocus sativus, is characterized by the presence of several apocarotenoids that contribute to the color, flavor, and aroma of the spice. However, little is known about the synthesis of aroma compounds during the development of the C. sativus stigma. The developing stigma is nearly odorless, but before and at anthesis, the aromatic compound beta-ionone becomes the principal norisoprenoid volatile in the stigma. In this study, four carotenoid cleavage dioxygenase (CCD) genes, CsCCD1a, CsCCD1b, CsCCD4a, and CsCCD4b, were isolated from C. sativus. Expression analysis showed that CsCCD1a was constitutively expressed, CsCCD1b was unique to the stigma tissue, but only CsCCD4a and -b had expression patterns consistent with the highest levels of beta-carotene and emission of beta-ionone derived during the stigma development. The CsCCD4 enzymes were localized in plastids and more specifically were present in the plastoglobules. The enzymatic activities of CsCCD1a, CsCCD1b, and CsCCD4 enzymes were determined by Escherichia coli expression, and subsequent analysis of the volatile products was generated by GC/MS. The four CCDs fell in two phylogenetically divergent dioxygenase classes, but all could cleave beta-carotene at the 9,10(9',10') positions to yield beta-ionone. The data obtained suggest that all four C. sativus CCD enzymes may contribute in different ways to the production of beta-ionone. In addition, the location and precise timing of beta-ionone synthesis, together with its known activity as a fragrance and insect attractant, suggest that this volatile may have a role in Crocus pollination.

  12. Redox proteins of hydroxylating bacterial dioxygenases establish a regulatory cascade that prevents gratuitous induction of tetralin biodegradation genes

    PubMed Central

    Ledesma-García, Laura; Sánchez-Azqueta, Ana; Medina, Milagros; Reyes-Ramírez, Francisca; Santero, Eduardo

    2016-01-01

    Bacterial dioxygenase systems are multicomponent enzymes that catalyze the initial degradation of many environmentally hazardous compounds. In Sphingopyxis granuli strain TFA tetralin dioxygenase hydroxylates tetralin, an organic contaminant. It consists of a ferredoxin reductase (ThnA4), a ferredoxin (ThnA3) and a oxygenase (ThnA1/ThnA2), forming a NAD(P)H–ThnA4–ThnA3–ThnA1/ThnA2 electron transport chain. ThnA3 has also a regulatory function since it prevents expression of tetralin degradation genes (thn) in the presence of non-metabolizable substrates of the catabolic pathway. This role is of physiological relevance since avoids gratuitous and wasteful production of catabolic enzymes. Our hypothesis for thn regulation implies that ThnA3 exerts its action by diverting electrons towards the regulator ThnY, an iron-sulfur flavoprotein that together with the transcriptional activator ThnR is necessary for thn gene expression. Here we analyze electron transfer among ThnA4, ThnA3 and ThnY by using stopped-flow spectrophotometry and determination of midpoint reduction potentials. Our results indicate that when accumulated in its reduced form ThnA3 is able to fully reduce ThnY. In addition, we have reproduced in vitro the regulatory circuit in the proposed physiological direction, NAD(P)H–ThnA4–ThnA3–ThnY. ThnA3 represents an unprecedented way of communication between a catabolic pathway and its regulatory system to prevent gratuitous induction. PMID:27030382

  13. Properties of catechol 1,2-dioxygenase in the cell free extract and immobilized extract of Mycobacterium fortuitum.

    PubMed

    Silva, A S; Jacques, R J S; Andreazza, R; Bento, F M; Roesch, L F W; Camargo, F A O

    2013-01-01

    Polycyclic aromatic hydrocarbons (PAH) are carcinogenic compounds which contaminate water and soil, and the enzymes can be used for bioremediation of these environments. This study aimed to evaluate some environmental conditions that affect the production and activity of the catechol 1,2-dioxygenase (C12O) by Mycobacterium fortuitum in the cell free and immobilized extract in sodium alginate. The bacterium was grown in mineral medium and LB broth containing 250 mg L(-1) of anthracene (PAH). The optimum conditions of pH (4.0-9.0), temperature (5-70 °C), reaction time (10-90 min) and the effect of ions in the enzyme activity were determined. The Mycobacterium cultivated in LB shown higher growth and the C12O activity was two-fold higher to that in the mineral medium. To both extracts the highest enzyme activity was at pH 8.0, however, the immobilized extract promoted the increase in the C12O activity in a pH range between 4.0 and 8.5. The immobilized extract increased the enzymatic activity time and showed the highest C12O activity at 45 °C, 20 °C higher than the greatest temperature in the cell free extract. The enzyme activity in both extracts was stimulated by Fe(3+), Hg(2+) and Mn(2+) and inhibited by NH(4+) and Cu(2+), but the immobilization protected the enzyme against the deleterious effects of K(+) and Mg(2+) in tested concentrations. The catechol 1,2-dioxygenase of Mycobacterium fortuitum in the immobilized extract has greater stability to the variations of pH, temperature and reaction time, and show higher activity in presence of ions, comparing to the cell free extract.

  14. Crystal Structure and Mechanism of Tryptophan 2,3-Dioxygenase, a Heme Enzyme Involved in Tryptophan Catabolism and in Quinolinate Biosynthesis

    SciTech Connect

    Zhang,Y.; Kang, S.; Mukherjee, T.; Bale, S.; Crane, B.; Begley, T.; Ealick, S.

    2007-01-01

    The structure of tryptophan 2,3-dioxygenase (TDO) from Ralstonia metallidurans was determined at 2.4 {angstrom}. TDO catalyzes the irreversible oxidation of L-tryptophan to N-formyl kynurenine, which is the initial step in tryptophan catabolism. TDO is a heme-containing enzyme and is highly specific for its substrate L-tryptophan. The structure is a tetramer with a heme cofactor bound at each active site. The monomeric fold, as well as the heme binding site, is similar to that of the large domain of indoleamine 2,3-dioxygenase, an enzyme that catalyzes the same reaction except with a broader substrate tolerance. Modeling of the putative (S)-tryptophan hydroperoxide intermediate into the active site, as well as substrate analogue and mutagenesis studies, are consistent with a Criegee mechanism for the reaction.

  15. Crystal Structure and Functional Analysis of the Extradiol Dioxygenase LapB from a Long-chain Alkylphenol Degradation Pathway in Pseudomonas*

    PubMed Central

    Cho, Jang-Hee; Jung, Du-Kyo; Lee, Kyoung; Rhee, Sangkee

    2009-01-01

    LapB is a non-heme Fe(II)-dependent 2,3-dioxygenase that catalyzes the second step of a long-chain alkylphenol (lap) degradation pathway in Pseudomonas sp. KL28 and belongs to the superfamily of type I extradiol dioxygenases. In this study, the crystal structures of substrate-free LapB and its complexes with a substrate or product were determined, along with a functional analysis of the active site residues. Structural features of the homotetramer are similar to those of other type I extradiol dioxygenases. In particular, the active site is located in the C-domain of each monomer, with a 2-His-1-carboxylate motif as the first coordination shell to iron ion. A comparison of three different structures in the catalytic cycle indicated catalysis-related local conformational changes in the active site. Specifically, the active site loop containing His-248 exhibits positional changes upon binding of the substrate and establishes a hydrogen-bonding network with Tyr-257, which is near the hydroxyl group of the substrate. Kinetic analysis of the mutant enzymes H248A, H248N, and Y257F showed that these three mutant enzymes are inactive, suggesting that this hydrogen-bonding network plays a crucial role in catalysis by deprotonating the incoming substrate and leaving it in a monoanionic state. Additional functional analysis of His-201, by using H201A and H201N mutants, near the dioxygen-binding site also supports its role as base and acid catalyst in the late stage of catalysis. We also noticed a disordered-to-ordered structural transition in the C-terminal region, resulting in the opening or closing of the active site. These results provide detailed insights into the structural and functional features of an extradiol dioxygenase that can accommodate a wide range of alkylcatechols. PMID:19828456

  16. Identification and Functional Analysis of Two Aromatic-Ring-Hydroxylating Dioxygenases from a Sphingomonas Strain That Degrades Various Polycyclic Aromatic Hydrocarbons

    PubMed Central

    Demanèche, Sandrine; Meyer, Christine; Micoud, Julien; Louwagie, Mathilde; Willison, John C.; Jouanneau, Yves

    2004-01-01

    In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [14C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases. PMID:15528538

  17. High activity catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 as a useful tool in cis,cis-muconic acid production.

    PubMed

    Guzik, Urszula; Hupert-Kocurek, Katarzyna; Sitnik, Małgorzata; Wojcieszyńska, Danuta

    2013-06-01

    This is the first report of a catechol 1,2-dioxygenase from Stenotrophomonas maltophilia strain KB2 with high activity against catechol and its methyl derivatives. This enzyme was maximally active at pH 8.0 and 40 °C and the half-life of the enzyme at this temperature was 3 h. Kinetic studies showed that the value of K m and V max was 12.8 μM and 1,218.8 U/mg of protein, respectively. During our studies on kinetic properties of the catechol 1,2-dioxygenase we observed substrate inhibition at >80 μM. The nucleotide sequence of the gene encoding the S. maltophilia strain KB2 catechol 1,2-dioxygenase has high identity with other catA genes from members of the genus Pseudomonas. The deduced 314-residue sequence of the enzyme corresponds to a protein of molecular mass 34.5 kDa. This enzyme was inhibited by competitive inhibitors (phenol derivatives) only by ca. 30 %. High tolerance against condition changes is desirable in industrial processes. Our data suggest that this enzyme could be of use as a tool in production of cis,cis-muconic acid and its derivatives.

  18. Fusion of Dioxygenase and Lignin-binding Domains in a Novel Secreted Enzyme from Cellulolytic Streptomyces sp. SirexAA-E*

    PubMed Central

    Bianchetti, Christopher M.; Harmann, Connor H.; Takasuka, Taichi E.; Hura, Gregory L.; Dyer, Kevin; Fox, Brian G.

    2013-01-01

    Streptomyces sp. SirexAA-E is a highly cellulolytic bacterium isolated from an insect/microbe symbiotic community. When grown on lignin-containing biomass, it secretes SACTE_2871, an aromatic ring dioxygenase domain fused to a family 5/12 carbohydrate-binding module (CBM 5/12). Here we present structural and catalytic studies of this novel fusion enzyme, thus providing insight into its function. The dioxygenase domain has the core β-sandwich fold typical of this enzyme family but lacks a dimerization domain observed in other intradiol dioxygenases. Consequently, the x-ray structure shows that the enzyme is monomeric and the Fe(III)-containing active site is exposed to solvent in a shallow depression on a planar surface. Purified SACTE_2871 catalyzes the O2-dependent intradiol cleavage of catechyl compounds from lignin biosynthetic pathways, but not their methylated derivatives. Binding studies show that SACTE_2871 binds synthetic lignin polymers and chitin through the interactions of the CBM 5/12 domain, representing a new binding specificity for this fold-family. Based on its unique structural features and functional properties, we propose that SACTE_2871 contributes to the invasive nature of the insect/microbial community by destroying precursors needed by the plant for de novo lignin biosynthesis as part of its natural wounding response. PMID:23653358

  19. The Mitochondrial Sulfur Dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 Is Required for Amino Acid Catabolism during Carbohydrate Starvation and Embryo Development in Arabidopsis1[C][W

    PubMed Central

    Krüßel, Lena; Junemann, Johannes; Wirtz, Markus; Birke, Hannah; Thornton, Jeremy D.; Browning, Luke W.; Poschet, Gernot; Hell, Rüdiger; Balk, Janneke; Braun, Hans-Peter; Hildebrandt, Tatjana M.

    2014-01-01

    The sulfur dioxygenase ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) catalyzes the oxidation of persulfides in the mitochondrial matrix and is essential for early embryo development in Arabidopsis (Arabidopsis thaliana). We investigated the biochemical and physiological functions of ETHE1 in plant metabolism using recombinant Arabidopsis ETHE1 and three transfer DNA insertion lines with 50% to 99% decreased sulfur dioxygenase activity. Our results identified a new mitochondrial pathway catalyzing the detoxification of reduced sulfur species derived from cysteine catabolism by oxidation to thiosulfate. Knockdown of the sulfur dioxygenase impaired embryo development and produced phenotypes of starvation-induced chlorosis during short-day growth conditions and extended darkness, indicating that ETHE1 has a key function in situations of high protein turnover, such as seed production and the use of amino acids as alternative respiratory substrates during carbohydrate starvation. The amino acid profile of mutant plants was similar to that caused by defects in the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase complex and associated dehydrogenases. Thus, in addition to sulfur amino acid catabolism, ETHE1 also affects the oxidation of branched-chain amino acids and lysine. PMID:24692429

  20. Acyclovir inhibits rat liver tryptophan-2,3-dioxygenase and induces a concomitant rise in brain serotonin and 5-hydroxyindole acetic acid levels.

    PubMed

    Müller, Adrienne C; Daya, Santy

    2008-09-01

    Viral diseases of the brain may induce changes in neurotransmitter synthesis and metabolism. In experimental herpes simplex encephalitis, brain serotonin is reduced, whilst it's major metabolite, 5-hydroxyindole acetic acid and turnover is increased. It is well established that reduced levels of brain monoamines, serotonin and norepinephrine may contribute to the symptoms of clinical depression, which raises the possibility that this condition is prevalent in herpes simplex encephalitis. An inverse relationship exists between liver tryptophan-2,3-dioxygenase activity and brain serotonin levels and there is an interdependency between serotonin and norepinephrine levels. The aim of this study is to determine the effect of acyclovir, an antiviral used in the treatment of herpes simplex encephalitis, on rat liver tryptophan-2,3-dioxygenase activity in vitro and in vivo as well as on rat forebrain serotonin, 5-hydroxyindole acetic acid and norepinephrine levels. The results show that acyclovir inhibits tryptophan-2,3-dioxygenase activity in vitro and in vivo, with a concomitant rise in serotonin and 5-hydroxyindole acetic acid levels. However, acyclovir reduces the turnover of serotonin to 5-hydroxyindole acetic acid, without any effect on norepinephrine levels. It appears that acyclovir may have the potential to reduce the clinical symptoms of depression in herpes simplex encephalitis. However, a greater turnover of serotonin to 5-hydroxyindole acetic acid could possibly be masked by conversion of serotonin to 5-hydroxytryptophol, which needs to be investigated further.

  1. Degradation potential of protocatechuate 3,4-dioxygenase from crude extract of Stenotrophomonas maltophilia strain KB2 immobilized in calcium alginate hydrogels and on glyoxyl agarose.

    PubMed

    Guzik, Urszula; Hupert-Kocurek, Katarzyna; Krysiak, Marta; Wojcieszyńska, Danuta

    2014-01-01

    Microbial intradiol dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5 °C and 10 °C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions.

  2. Analysis of PCBs degradation abilities of biphenyl dioxygenase derived from Enterobacter sp. LY402 by molecular simulation.

    PubMed

    Cao, Ya-Ming; Xu, Li; Jia, Ling-Yun

    2011-12-15

    Enterobacter sp. LY402 is a bacterium isolated from polluted soil. It can efficiently degrade polychlorinated biphenyls (PCBs) under aerobic conditions. However, the degradation was limited when it comes to high chlorine or double para-substituted PCBs. Biphenyl dioxygenase (BDO) is the key enzyme in the PCBs biodegradation process. It has been confirmed that the α-subunit of the iron-sulfur protein of biphenyl 2,3-dioxygenase (BphA1) directly influenced catalytic activities and substrate specificity. To know the degradation characteristics of BDO to PCBs, we analyzed PCBs degradation abilities of BphA1 from Enterobacter sp. LY402 by experiment and molecular simulation. Firstly, the degradation experiment of PCBs was performed, and the degradation rate constants (k) were calculated. Then the three-dimensional model of LY402-BphA1 was constructed. Through further docking studies with 209 PCB congeners, the PCBs binding abilities of LY402-BphA1 were measured and some crucial active site residues were identified. Moreover, the molecular descriptors of PCBs were calculated and analyzed to determine the correlation of molecular properties and degradation. The results showed that the affinity energy of PCBs was well matched with the k values of the different number of chlorine substituents. The binding ability of BphA1 greatly affected the PCBs degradation abilities of BDO. Hydrophobic contact was the main interaction between the residues of active site and PCB substrates. The number and subposition of chlorine substituents would influence the PCBs binding ability of BphA1 significantly. Ser283, Val287, Gly321 and Tyr384 residues in the active site of LY402-BphA1 showed high variability, and the space limitation of the active site of BphA1 had negative influence on the PCBs binding affinity of BDO. The changes of physical, electronic and geometrical properties could influence degradation and binding affinity of PCBs. Analysis of structural information, binding affinity

  3. The carotenoid cleavage dioxygenase CCD2 catalysing the synthesis of crocetin in spring crocuses and saffron is a plastidial enzyme.

    PubMed

    Ahrazem, Oussama; Rubio-Moraga, Angela; Berman, Judit; Capell, Teresa; Christou, Paul; Zhu, Changfu; Gómez-Gómez, Lourdes

    2016-01-01

    The apocarotenoid crocetin and its glycosylated derivatives, crocins, confer the red colour to saffron. Crocetin biosynthesis in saffron is catalysed by the carotenoid cleavage dioxygenase CCD2 (AIG94929). No homologues have been identified in other plant species due to the very limited presence of crocetin and its derivatives in the plant kingdom. Spring Crocus species with yellow flowers accumulate crocins in the stigma and tepals. Four carotenoid CCDs, namely CaCCD1, CaCCD2 and CaCCD4a/b and CaCCD4c were first cloned and characterized. CaCCD2 was localized in plastids, and a longer CCD2 version, CsCCD2L, was also localized in this compartment. The activity of CaCCD2 was assessed in Escherichia coli and in a stable rice gene function characterization system, demonstrating the production of crocetin in both systems. The expression of all isolated CCDs was evaluated in stigma and tepals at three key developmental stages in relation with apocarotenoid accumulation. CaCCD2 expression parallels crocin accumulation, but C14 apocarotenoids most likely are associated to the CaCCD1 activity in Crocus ancyrensis flowers. The specific CCD2 localization and its membrane interaction will contribute to the development of a better understanding of the mechanism of crocetin biosynthesis and regulation in the chromoplast.

  4. Transgenic Leucaena leucocephala expressing the Rhizobium gene pydA encoding a meta-cleavage dioxygenase shows reduced mimosine content.

    PubMed

    Jube, Sandro L R; Borthakur, Dulal

    2010-04-01

    The use of the tree-legume Leucaena leucocephala (leucaena), which contains high levels of proteins in its foliage, is limited due to the presence of the toxic free amino acid mimosine. The goal of this research was to develop transgenic leucaena with reduced mimosine content. Two genes, pydA and pydB, encoding a meta-cleavage dioxygenase (EC 1.13.11.2) and a pyruvate hydrolase (EC 3.7.1.6), respectively, from the mimosine-degrading leucaena symbiont Rhizobium sp. strain TAL1145, were used to transform leucaena. These bacterial genes were sequence-optimized for expression in leucaena and cloned into the plant binary vector pCAMBIA3201 for Agrobacterium tumefaciens-mediated transformation. Using immature zygotic embryos as the start explant material, six pydA and three pydB transgenic lines were developed. The presence and expression of the bacterial genes in the transgenic lines were verified by PCR, reverse transcriptase PCR, and Southern analyses. HPLC analyses of the transgenic plants determined that the mimosine contents of the pydA-expressing lines were reduced up to 22.5% in comparison to the wild-type. No significant reduction in mimosine content was observed in the pydB-expressing lines. This is the first example of using a gene from a bacterial symbiont to reduce the toxicity of a tree-legume.

  5. Unity in diversity, a systems approach to regulating plant cell physiology by 2-oxoglutarate-dependent dioxygenases.

    PubMed

    Kundu, Siddhartha

    2015-01-01

    Could a disjoint group of enzymes synchronize their activities and execute a complex multi-step, measurable, and reproducible response? Here, I surmise that the alpha-ketoglutarate dependent superfamily of non-haem iron (II) dioxygenases could influence cell physiology as a cohesive unit, and that the broad spectra of substrates transformed is an absolute necessity to this portrayal. This eclectic group comprises members from all major taxa, and participates in pesticide breakdown, hypoxia signaling, and osmotic stress neutralization. The oxidative decarboxylation of 2-oxoglutarate to succinate is coupled with a concomitant substrate hydroxylation and, in most cases, is followed by an additional specialized conversion. The domain profile of a protein sequence was used as an index of miscellaneous reaction chemistry and interpreted alongside existent kinetic data in a linear model of integrated function. Statistical parameters were inferred by the creation of a novel, empirically motivated flat-file database of over 3800 sequences (DB2OG) with putative 2-oxoglutarate dependent activity. The collated information was categorized on the basis of existing annotation schema. The data suggests that 2OG-dependent enzymes incorporate several desirable features of a systems level player. DB2OG, is free, accessible without a login to all users, and available at the following URL (http://comp-biol.theacms.in/DB2OG.html).

  6. Docking studies of antidepressants against single crystal structure of tryptophan 2, 3-dioxygenase using Molegro Virtual Docker software.

    PubMed

    Dawood, Shazia; Zarina, Shamshad; Bano, Samina

    2014-09-01

    Tryptophan 2, 3-dioxygenase (TDO) a heme containing enzyme found in mammalian liver is responsible for tryptophan (Trp) catabolism. Trp is an essential amino acid that is degraded in to N-formylkynurenine by the action of TDO. The protein ligand interaction plays a significant role in structural based drug designing. The current study illustrates the binding of established antidepressants (ADs) against TDO enzyme using in-silico docking studies. For this purpose, Fluoxetine, Paroxetine, Sertraline, Fluvoxamine, Seproxetine, Citalopram, Moclobamide, Hyperforin and Amoxepine were selected. In-silico docking studies were carried out using Molegro Virtual Docker (MVD) software. Docking results show that all ADs fit well in the active site of TDO moreover Hyperforin and Paroxetine exhibited high docking scores of -152.484k cal/mol and -139.706k cal/mol, respectively. It is concluded that Hyperforin and Paroxetine are possible lead molecules because of their high docking scores as compared to other ADs examined. Therefore, these two ADs stand as potent inhibitors of TDO enzyme.

  7. Refining the reaction mechanism of O2 towards its co-substrate in cofactor-free dioxygenases

    PubMed Central

    2016-01-01

    Cofactor-less oxygenases perform challenging catalytic reactions between singlet co-substrates and triplet oxygen, in spite of apparently violating the spin-conservation rule. In 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase, the active site has been suggested by quantum chemical computations to fine tune triplet oxygen reactivity, allowing it to interact rapidly with its singlet substrate without the need for spin inversion, and in urate oxidase the reaction is thought to proceed through electron transfer from the deprotonated substrate to an aminoacid sidechain, which then feeds the electron to the oxygen molecule. In this work, we perform additional quantum chemical computations on these two systems to elucidate several intriguing features unaddressed by previous workers. These computations establish that in both enzymes the reaction proceeds through direct electron transfer from co-substrate to O2 followed by radical recombination, instead of minimum-energy crossing points between singlet and triplet potential energy surfaces without formal electron transfer. The active site does not affect the reactivity of oxygen directly but is crucial for the generation of the deprotonated form of the co-substrates, which have redox potentials far below those of their protonated forms and therefore may transfer electrons to oxygen without sizeable thermodynamic barriers. This mechanism seems to be shared by most cofactor-less oxidases studied so far. PMID:28028471

  8. Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets.

    PubMed

    White, Mark D; Klecker, Maria; Hopkinson, Richard J; Weits, Daan A; Mueller, Carolin; Naumann, Christin; O'Neill, Rebecca; Wickens, James; Yang, Jiayu; Brooks-Bartlett, Jonathan C; Garman, Elspeth F; Grossmann, Tom N; Dissmeyer, Nico; Flashman, Emily

    2017-03-23

    Crop yield loss due to flooding is a threat to food security. Submergence-induced hypoxia in plants results in stabilization of group VII ETHYLENE RESPONSE FACTORs (ERF-VIIs), which aid survival under these adverse conditions. ERF-VII stability is controlled by the N-end rule pathway, which proposes that ERF-VII N-terminal cysteine oxidation in normoxia enables arginylation followed by proteasomal degradation. The PLANT CYSTEINE OXIDASEs (PCOs) have been identified as catalysts of this oxidation. ERF-VII stabilization in hypoxia presumably arises from reduced PCO activity. We directly demonstrate that PCO dioxygenase activity produces Cys-sulfinic acid at the N terminus of an ERF-VII peptide, which then undergoes efficient arginylation by an arginyl transferase (ATE1). This provides molecular evidence of N-terminal Cys-sulfinic acid formation and arginylation by N-end rule pathway components, and a substrate of ATE1 in plants. The PCOs and ATE1 may be viable intervention targets to stabilize N-end rule substrates, including ERF-VIIs, to enhance submergence tolerance in agriculture.

  9. CAROTENOID CLEAVAGE DIOXYGENASE4 Is a Negative Regulator of β-Carotene Content in Arabidopsis Seeds[W

    PubMed Central

    Gonzalez-Jorge, Sabrina; Ha, Sun-Hwa; Magallanes-Lundback, Maria; Gilliland, Laura Ullrich; Zhou, Ailing; Lipka, Alexander E.; Nguyen, Yen-Nhu; Angelovici, Ruthie; Lin, Haining; Cepela, Jason; Little, Holly; Buell, C. Robin; Gore, Michael A.; DellaPenna, Dean

    2013-01-01

    Experimental approaches targeting carotenoid biosynthetic enzymes have successfully increased the seed β-carotene content of crops. However, linkage analysis of seed carotenoids in Arabidopsis thaliana recombinant inbred populations showed that only 21% of quantitative trait loci, including those for β-carotene, encode carotenoid biosynthetic enzymes in their intervals. Thus, numerous loci remain uncharacterized and underutilized in biofortification approaches. Linkage mapping and genome-wide association studies of Arabidopsis seed carotenoids identified CAROTENOID CLEAVAGE DIOXYGENASE4 (CCD4) as a major negative regulator of seed carotenoid content, especially β-carotene. Loss of CCD4 function did not affect carotenoid homeostasis during seed development but greatly reduced carotenoid degradation during seed desiccation, increasing β-carotene content 8.4-fold relative to the wild type. Allelic complementation of a ccd4 null mutant demonstrated that single-nucleotide polymorphisms and insertions and deletions at the locus affect dry seed carotenoid content, due at least partly to differences in CCD4 expression. CCD4 also plays a major role in carotenoid turnover during dark-induced leaf senescence, with β-carotene accumulation again most strongly affected in the ccd4 mutant. These results demonstrate that CCD4 plays a major role in β-carotene degradation in drying seeds and senescing leaves and suggest that CCD4 orthologs would be promising targets for stabilizing and increasing the level of provitamin A carotenoids in seeds of major food crops. PMID:24368792

  10. Molecular Modeling and Dynamic Simulation of Arabidopsis thaliana Carotenoid Cleavage Dioxygenase Gene: A Comparison with Bixa orellana and Crocus sativus.

    PubMed

    R, Priya; P, Sneha; Madrid, Renata Rivera; C, George Priya Doss; Singh, Pooja; Ramamoorthy, Siva

    2017-02-01

    Carotenoid cleavage dioxygenase (CCD) gene, ubiquitously found in numerous types of plants, are eminent in synthesizing the various volatile compounds (β-ionone, C13 -norisoprenoid, geranylacetone) known as apocarotenoids. These apocarotenoids have various biological functions such as volatile signals, allelopathic interaction and plant defense. In Arabidopsis genome sequence, four potential CCD genes have been identified namely CCD1, CCD4, CCD7, and CCD8. These four genes give rise to diverse biological functions with almost similar sequence identity. In this investigation, an in silico analysis was proposed to study CCD proteins in Arabidopsis thaliana, aiming at constructing three-dimensional (3D) structure for CCD1 proteins of Bixa orellana and Crocus sativus to observe the structural difference among AtCCD (A. thaliana CCD) proteins. The quality of modeled structures was evaluated using RAMPAGE, PSVS protein validation server and Q Mean server. Finally, we utilised molecular dynamics simulation to identify the stability of the predicted CCD protein structures. The molecular dynamic simulation also revealed that AtCCD4 protein showed lesser stability when compared to other CCD proteins. Overall results from molecular dynamics analysis predicted that BoCCD1, CsCCD1, and AtCCD1 show similar structural characteristics. This article is protected by copyright. All rights reserved.

  11. Metal-dependent activity of Fe and Ni acireductone dioxygenases: how two electrons reroute the catalytic pathway.

    PubMed

    Sparta, Manuel; Valdez, Crystal E; Alexandrova, Anastassia N

    2013-08-23

    Two virtually identical acireductone dioxygenases, ARD and ARD', catalyze completely different oxidation reactions of the same substrate, 1,2-dihydroxy-3-keto-5-(methylthio)pentene, depending exclusively on the nature of the bound metal. Fe(2+)-dependent ARD' produces the α-keto acid precursor of methionine and formate and allows for the recycling of methionine in cells. Ni(2+)-dependent ARD instead produces methylthiopropionate, CO, and formate, and exits the methionine salvage cycle. This mechanistic difference has not been understood to date but has been speculated to be due to the difference in coordination of the substrate to Fe(2+)versus Ni(2+): forming a five-membered ring versus a six-membered ring, respectively, thus exposing different carbon atoms for the attack by O2. Here, using mixed quantum-classical molecular dynamics simulations followed by the density functional theory mechanistic investigation, we show that, contrary to the old hypothesis, both metals preferentially bind the substrate as a six-membered ring, exposing the exact same sites to the attack by O2. It is the electronic properties of the metals that are then responsible for the system following different reaction paths, to yield the respective products. We fully explain the puzzling metal-induced difference in functionality between ARD and ARD' and, in particular, propose a new mechanism for ARD'. All results are in agreement with available isotopic substitution and other experimental data.

  12. Arabidopsis ETHE1 Encodes a Sulfur Dioxygenase That Is Essential for Embryo and Endosperm Development1[C][OA

    PubMed Central

    Holdorf, Meghan M.; Owen, Heather A.; Lieber, Sarah Rhee; Yuan, Li; Adams, Nicole; Dabney-Smith, Carole; Makaroff, Christopher A.

    2012-01-01

    Mutations in human (Homo sapiens) ETHYLMALONIC ENCEPHALOPATHY PROTEIN1 (ETHE1) result in the complex metabolic disease ethylmalonic encephalopathy, which is characterized in part by brain lesions, lactic acidemia, excretion of ethylmalonic acid, and ultimately death. ETHE1-like genes are found in a wide range of organisms; however, the biochemical and physiological role(s) of ETHE1 have not been examined outside the context of ethylmalonic encephalopathy. In this study we characterized Arabidopsis (Arabidopsis thaliana) ETHE1 and determined the effect of an ETHE1 loss-of-function mutation to investigate the role(s) of ETHE1 in plants. Arabidopsis ETHE1 is localized in the mitochondrion and exhibits sulfur dioxygenase activity. Seeds homozygous for a DNA insertion in ETHE1 exhibit alterations in endosperm development that are accompanied by a delay in embryo development followed by embryo arrest by early heart stage. Strong ETHE1 labeling was observed in the peripheral and chalazal endosperm of wild-type seeds prior to cellularization. Therefore, ETHE1 appears to play an essential role in regulating sulfide levels in seeds. PMID:22786886

  13. Indoleamine 2,3-Dioxygenase Is Dispensable for The Immunomodulatory Function of Stem Cells from Human Exfoliated Deciduous Teeth

    PubMed Central

    Alipour, Razieh; Masoumi Karimi, Masoumeh; Hashemi-Beni, Batool; Adib, Minoo; Sereshki, Nasrin; Sadeghi, Farzaneh

    2017-01-01

    Objective In this study, we sought to better understand the immunoregulatory function of stem cells derived from human exfoliated deciduous teeth (SHED). We studied the role of the interferon gamma (IFN-γ)-indoleamine 2,3-dioxygenase (IDO)-axis in immunoregulation of SHED compared to bone marrow derived mesenchymal stem cells (BMMSCs) under the same conditions. Materials and Methods In this cross-sectional study, recently isolated human T cells were stimulated either by mitogen or inactivated allogeneic peripheral blood mononuclear cells (PBMCs). These T cells were subsequently co-cultured with, either SHED or BMMSCs in the presence or absence of 1-methyl-tryptophan (1-MT) or neutralizing anti- human-IFN-γ antibodies. In all co-cultures we evaluated lymphocyte activation as well as IDO activity. Results SHED, similar to conventional BMMSCs, had anti-proliferative effects on stimulated T cells and reduced their cytokine production. This property of SHED and BMMSCs was changed by IFN-γ neutralization. We detected IDO in the immunosuppressive supernatant of all co-cultures. Removal of IDO decreased the immunosuppression of BMMSCs. Conclusion SHED, like BMMSCs, produced the IDO enzyme. Although IFN-γ is one of inducer of IDO production in SHED, these cells were not affected by IFN-γ in the same manner as BMMSCs. Unlike BMMSCs, the IDO enzyme did not contribute to their immunosuppression and might have other cell-type specific roles. PMID:28042544

  14. Putative mitochondrial α-ketoglutarate-dependent dioxygenase Fmp12 controls utilization of proline as an energy source in Saccharomyces cerevisiae

    PubMed Central

    Nishida, Ikuhisa; Watanabe, Daisuke; Takagi, Hiroshi

    2016-01-01

    The amino acid proline functions as a nitrogen source and as a stress protectant in the yeast Saccharomyces cerevisiae. However, utilization of proline as a carbon source in S. cerevisiae cells has not been studied yet. In the process of study on the physiological roles of the found-in-mitochondrial-proteome (FMP) genes in proline metabolism, we found that Δfmp12 cells could grow better than wild-type cells on agar plate medium containing proline as the sole nitrogen and carbon sources. In contrast, overexpression of FMP12 negatively affected cell growth under the same condition. The Fmp12 protein was localized in the mitochondria and was constitutively expressed. Deletion of the genes that encode mitochondrial enzymes, such as proline dehydrogenase (PUT1), Δ1-pyrroline-5-carboxylate dehydrogenase (PUT2), alanine transaminase (ALT1), and α-ketoglutarate dehydrogenase subunit (KGD1), abolished the enhanced cell growth in Δfmp12. These results provided the first evidence that proline can be utilized as a carbon source via the mitochondrial proline metabolic pathway and the subsequent tricarboxylic acid (TCA) cycle in S. cerevisiae. The function of Fmp12, which has a similarity with α-ketoglutarate-dependent dioxygenases of the yeast Candida species and human, might inhibit cell growth by skipping the ATP production step of the TCA cycle. PMID:28357320

  15. Beta-triketone inhibitors of plant p-hydroxyphenylpyruvate dioxygenase: modeling and comparative molecular field analysis of their interactions.

    PubMed

    Dayan, Franck E; Singh, Nidhi; McCurdy, Christopher R; Godfrey, Colette A; Larsen, Lesley; Weavers, Rex T; Van Klink, John W; Perry, Nigel B

    2009-06-24

    p-Hydroxyphenylpyruvate dioxygenase (HPPD) is the target site of beta-triketone herbicides in current use. Nineteen beta-triketones and analogues, including the naturally occurring leptospermone and grandiflorone, were synthesized and tested as inhibitors of purified Arabidopsis thaliana HPPD. The most active compound was a beta-triketone with a C(9) alkyl side chain, not reported as natural, which inhibited HPPD with an I(50) of 19 +/- 1 nM. This is significantly more active than sulcotrione, which had an I(50) of 250 +/- 21 nM in this assay system. The most active naturally occurring beta-triketone was grandiflorone, which had an I(50) of 750 +/- 70 nM. This compound is of potential interest as a natural herbicide because it can be extracted with good yield and purity from some Leptospermum shrubs. Analogues without the 1,3-diketone group needed to interact with Fe(2+) at the HPPD active site were inactive (I(50)s > 50 microM), as were analogues with prenyl or ethyl groups on the triketone ring. Modeling of the binding of the triketones to HPPD, three-dimensional QSAR analysis using CoMFA (comparative molecular field analysis), and evaluation of the hydrophobic contribution with HINT (hydropathic interactions) provided a structural basis to describe the ligand/receptor interactions.

  16. Disruption of a CAROTENOID CLEAVAGE DIOXYGENASE 4 gene converts flower colour from white to yellow in Brassica species.

    PubMed

    Zhang, Bao; Liu, Chao; Wang, Yaqin; Yao, Xuan; Wang, Fang; Wu, Jiangsheng; King, Graham J; Liu, Kede

    2015-06-01

    In Brassica napus, yellow petals had a much higher content of carotenoids than white petals present in a small number of lines, with violaxanthin identified as the major carotenoid compound in yellow petals of rapeseed lines. Using positional cloning we identified a carotenoid cleavage dioxygenase 4 gene, BnaC3.CCD4, responsible for the formation of flower colour, with preferential expression in petals of white-flowered B. napus lines. Insertion of a CACTA-like transposable element 1 (TE1) into the coding region of BnaC3.CCD4 had disrupted its expression in yellow-flowered rapeseed lines. α-Ionone was identified as the major volatile apocarotenoid released from white petals but not from yellow petals. We speculate that BnaC3.CCD4 may use δ- and/or α-carotene as substrates. Four variations, including two CACTA-like TEs (alleles M1 and M4) and two insertion/deletions (INDELs, alleles M2 and M3), were identified in yellow-flowered Brassica oleracea lines. The two CACTA-like TEs were also identified in the coding region of BcaC3.CCD4 in Brassica carinata. However, the two INDELs were not detected in B. napus and B. carinata. We demonstrate that the insertions of TEs in BolC3.CCD4 predated the formation of the two allotetraploids.

  17. Local gene therapy with indoleamine 2,3-dioxygenase protects against development of transplant vasculopathy in chronic kidney transplant dysfunction.

    PubMed

    Vavrincova-Yaghi, D; Deelman, L E; van Goor, H; Seelen, M A; Vavrinec, P; Kema, I P; Gomolcak, P; Benigni, A; Henning, R H; Sandovici, M

    2016-11-01

    Chronic transplant dysfunction (CTD) is the primary cause of late allograft loss in kidney transplantation. Indoleamine 2,3-dioxygenase (IDO) is involved in fetomaternal tolerance and IDO gene therapy inhibits acute rejection following kidney transplantation. The aim of this study is to investigate whether gene therapy with IDO is able to attenuate CTD. Transplantation was performed in a rat Dark-Agouti to Wistar-Furth CTD model. Donor kidneys were incubated either with an adenovirus carrying IDO gene, a control adenovirus or saline. During the first 10 days recipients received low-dose cyclosporine. Body weight, blood pressure, serum creatinine and proteinuria were measured every 2 weeks. Rats were killed after 12 weeks. IDO had a striking beneficial effect on transplant vasculopathy at week 12. It also significantly improved body weight gain; it reduced blood pressure and decreased proteinuria during the follow-up. However, it did not affect the kidney function. In addition, IDO therapy significantly decreased the number of graft-infiltrating macrophages at week 12. The messenger RNA levels of forkhead box p3 and transforming grow factor-β were elevated in the IDO treated group at week 12. Here we show for first time a clear beneficial effect of local IDO gene therapy especially on transplant vasculopathy in a rat model of renal CTD.

  18. ABA-mediated heterophylly is regulated by differential expression of 9-cis-epoxycarotenoid dioxygenase 3 in lilies.

    PubMed

    Chen, Hung-Chi; Hwang, San-Gwang; Chen, Shiau-Ming; Shii, Chou-Tou; Cheng, Wan-Hsing

    2011-10-01

    Although exogenous ABA-regulated heterophylly has been well documented in multiple plant species, the effect of endogenous ABA and its molecular mechanism remain uncharacterized. In the present study, the effects of endogenous ABA on heterophyllous switching were investigated in two different lily varieties, Lilium formosanum and Lilium oriental hybrid 'Casa Blanca'. Seedlings of L. formosanum, which have scale-leaf-type growth, displayed low levels of both 9-cis-epoxycarotenoid dioxygenase 3 (LfNCED3) transcripts and ABA, whereas seedlings of L. oriental hybrid 'Casa Blanca', which have scale-type growth, displayed high levels of both LoNCED3 transcripts and ABA. Sucrose induced endogenous ABA production in cultured lilies; low ABA induction shows scale-leaf-type growth, whereas scale-type growth becomes predominant when ABA levels are high. Heterologous expression of either LfNCED3 or LoNCED3 was found to complement the Arabidopsis Atnced3 mutant. Interestingly, the expression patterns of LfNCED3 and LoNCED3 in transgenic Arabidopsis plants are distinguishable. Further promoter analysis revealed that a putative E2F-like element in the LfNCED3 promoter, but not in the LoNCED3 promoter, plays a negative role in controlling its activity. Collectively, our results demonstrate that NCED3 plays a key role in ABA-mediated heterophylly in lilies.

  19. Structural Characterization of Pandoraea pnomenusa B-356 Biphenyl Dioxygenase Reveals Features of Potent Polychlorinated Biphenyl-Degrading Enzymes

    PubMed Central

    Chakko, Mathew N.; Sinha, Sangita C.; Powlowski, Justin B.; Eltis, Lindsay D.; Bolin, Jeffrey T.

    2013-01-01

    The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase (BPDOB356). BPDOB356, a heterohexameric (αβ)3 Rieske oxygenase (RO), catalyzes the insertion of dioxygen with stereo- and regioselectivity at the 2,3-carbons of biphenyl, and can transform a broad spectrum of PCB congeners. Here we present the X-ray crystal structures of BPDOB356 with and without its substrate biphenyl 1.6-Å resolution for both structures. In both cases, the Fe(II) has five ligands in a square pyramidal configuration: H233 Nε2, H239 Nε2, D386 Oδ1 and Oδ2, and a single water molecule. Analysis of the active sites of BPDOB356 and related ROs revealed structural features that likely contribute to the superior PCB-degrading ability of certain BPDOs. First, the active site cavity readily accommodates biphenyl with minimal conformational rearrangement. Second, M231 was predicted to sterically interfere with binding of some PCBs, and substitution of this residue yielded variants that transform 2,2′-dichlorobiphenyl more effectively. Third, in addition to the volume and shape of the active site, residues at the active site entrance also apparently influence substrate preference. Finally, comparison of the conformation of the active site entrance loop among ROs provides a basis for a structure-based classification consistent with a phylogeny derived from amino acid sequence alignments. PMID:23308114

  20. Structure prediction of Fe(II) 2-oxoglutarate dioxygenase from a psychrophilic yeast Glaciozyma antarctica PI12

    NASA Astrophysics Data System (ADS)

    Yusof, Nik Yusnoraini; Bakar, Farah Diba Abu; Mahadi, Nor Muhammad; Raih, Mohd Firdaus; Murad, Abdul Munir Abdul

    2015-09-01

    A cDNA encoding Fe(II) 2-oxoglutarate (2OG) dependent dioxygenases was isolated from psychrophilic yeast, Glaciozyma antarctica PI12. We have successfully amplified 1,029 bp cDNA sequence that encodes 342 amino acid with predicted molecular weight 38 kDa. The prediction protein was analysed using various bioinformatics tools to explore the properties of the protein. Based on a BLAST search analysis, the Fe2OX amino acid sequence showed 61% identity to the sequence of oxoglutarate/iron-dependent oxygenase from Rhodosporidium toruloides NP11. SignalP prediction showed that the Fe2OX protein contains no putative signal peptide, which suggests that this enzyme most probably localised intracellularly.The structure of Fe2OX was predicted by homology modelling using MODELLER9v11. The model with the lowest objective function was selected from hundred models generated using MODELLER9v11. Analysis of the structure revealed the longer loop at Fe2OX from G.antarctica that might be responsible for the flexibility of the structure, which contributes to its adaptation to low temperatures. Fe2OX hold a highly conserved Fe(II) binding HXD/E…H triad motif. The binding site for 2-oxoglutarate was found conserved for Arg280 among reported studies, however the Phe268 was found to be different in Fe2OX.

  1. P-HYDROXYPHENYLPYRUVATE DIOXYGENASE from Medicago sativa is involved in vitamin E biosynthesis and abscisic acid-mediated seed germination

    PubMed Central

    Jiang, Jishan; Chen, Zhihong; Ban, Liping; Wu, Yudi; Huang, Jianping; Chu, Jinfang; Fang, Shuang; Wang, Zan; Gao, Hongwen; Wang, Xuemin

    2017-01-01

    P-HYDROXYPHENYLPYRUVATE DIOXYGENASE (HPPD) is the first committed enzyme involved in the biosynthesis of vitamin E, and is characterized by catalyzing the conversion of p-hydroxyphenyl pyruvate (HPP) to homogentisic acid (HGA). Here, an HPPD gene was cloned from Medicago sativa L. and designated MsHPPD, which was expressed at high levels in alfalfa leaves. PEG 6000 (polyethylene glycol), NaCl, abscisic acid and salicylic acid were shown to significantly induce MsHPPD expression, especially in the cotyledons and root tissues. Overexpression of MsHPPD was found to significantly increase the level of β-tocotrienol and the total vitamin E content in Arabidopsis seeds. Furthermore, these transgenic Arabidopsis seeds exhibited an accelerated germination time, compared with wild-type seeds under normal conditions, as well as under NaCl and ABA treatments. Meanwhile, the expression level of several genes associated with ABA biosynthesis (NCED3, NCED5 and NCED9) and the ABA signaling pathway (RAB18, ABI3 and ABI5) were significantly down-regulated in MsHPPD-overexpressing transgenic lines, as well as the total free ABA content. Taken together, these results demonstrate that MsHPPD functions not only in the vitamin E biosynthetic pathway, but also plays a critical role in seed germination via affecting ABA biosynthesis and signaling. PMID:28084442

  2. CAROTENOID CLEAVAGE DIOXYGENASE 7 modulates plant growth, reproduction, senescence, and determinate nodulation in the model legume Lotus japonicus

    PubMed Central

    Liu, Junwei; Novero, Mara; Charnikhova, Tatsiana; Ferrandino, Alessandra; Schubert, Andrea; Ruyter-Spira, Carolien; Bonfante, Paola; Lovisolo, Claudio; Bouwmeester, Harro J.; Cardinale, Francesca

    2013-01-01

    Strigolactones (SLs) are newly identified hormones that regulate multiple aspects of plant development, infection by parasitic weeds, and mutualistic symbiosis in the roots. In this study, the role of SLs was studied for the first time in the model plant Lotus japonicus using transgenic lines silenced for CAROTENOID CLEAVAGE DIOXYGENASE 7 (LjCCD7), the orthologue of Arabidopsis More Axillary Growth 3. Transgenic LjCCD7-silenced plants displayed reduced height due to shorter internodes, and more branched shoots and roots than the controls, and an increase in total plant biomass, while their root:shoot ratio remained unchanged. Moreover, these lines had longer primary roots, delayed senescence, and reduced flower/pod numbers from the third round of flower and pod setting onwards. Only a mild reduction in determinate nodule numbers and hardly any impact on the colonization by arbuscular mycorrhizal fungi were observed. The results show that the impairment of CCD7 activity in L. japonicus leads to a phenotype linked to SL functions, but with specific features possibly due to the peculiar developmental pattern of this plant species. It is believed that the data also link determinate nodulation, plant reproduction, and senescence to CCD7 function for the first time. PMID:23567864

  3. New insight into the cleavage reaction of Nostoc sp. strain PCC 7120 carotenoid cleavage dioxygenase in natural and nonnatural carotenoids.

    PubMed

    Heo, Jinsol; Kim, Se Hyeuk; Lee, Pyung Cheon

    2013-06-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8'-carotenal at 3 positions, C-13 C-14, C-15 C-15', and C-13' C-14', revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4'-diaponeurosporene, 4,4'-diaponeurosporen-4'-al, 4,4'-diaponeurosporen-4'-oic acid, 4,4'-diapotorulene, and 4,4'-diapotorulen-4'-al to generate novel cleavage products (apo-14'-diaponeurosporenal, apo-13'-diaponeurosporenal, apo-10'-diaponeurosporenal, apo-14'-diapotorulenal, and apo-10'-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro.

  4. Lack of congruence between cysteine dioxygenase activity and S-carboxymethyl-L-cysteine S-oxidation activity in rat cytosol.

    PubMed

    Khan, Samera; Mitchell, Stephen C; Steventon, Glyn B

    2004-08-01

    The identity of the enzyme(s) responsible for the S-oxidation of the mucoactive drug S-carboxymethyl-L-cysteine (SCMC) is unknown but the protein(s) are a susceptibility factor for a number of chronic degenerative diseases. The structural similarities between the amino acid L-cysteine and SCMC have raised the possibility that cysteine dioxygenase (CDO) may be responsible for this biotransformation reaction. Both CDO and SCMC S-oxygenase were found to require Fe2+ for enzymatic activity, and both enzyme activities were inhibited by Fe2+ and Fe3+ chelators. However, sulphydryl group modification of the enzymes resulted in the activation of the S-oxidation of SCMC but inhibition of the S-oxidation of L-cysteine. When the two enzyme activities were quantified in 20 female hepatic cytosolic fractions no linear correlation in the production of their respective metabolites was seen. The results of this investigation indicate that CDO is not responsible for the S-oxidation of SCMC in the rat.

  5. Dioxygenases catalyze O-demethylation and O,O-demethylenation with widespread roles in benzylisoquinoline alkaloid metabolism in opium poppy.

    PubMed

    Farrow, Scott C; Facchini, Peter J

    2013-10-04

    In opium poppy, the antepenultimate and final steps in morphine biosynthesis are catalyzed by the 2-oxoglutarate/Fe(II)-dependent dioxygenases, thebaine 6-O-demethylase (T6ODM) and codeine O-demethylase (CODM). Further investigation into the biochemical functions of CODM and T6ODM revealed extensive and unexpected roles for such enzymes in the metabolism of protopine, benzo[c]phenanthridine, and rhoeadine alkaloids. When assayed with a wide range of benzylisoquinoline alkaloids, CODM, T6ODM, and the functionally unassigned paralog DIOX2, renamed protopine O-dealkylase, showed novel and efficient dealkylation activities, including regio- and substrate-specific O-demethylation and O,O-demethylenation. Enzymes catalyzing O,O-demethylenation, which cleave a methylenedioxy bridge leaving two hydroxyl groups, have previously not been reported in plants. Similar cleavage of methylenedioxy bridges on substituted amphetamines is catalyzed by heme-dependent cytochromes P450 in mammals. Preferred substrates for O,O-demethylenation by CODM and protopine O-dealkylase were protopine alkaloids that serve as intermediates in the biosynthesis of benzo[c]phenanthridine and rhoeadine derivatives. Virus-induced gene silencing used to suppress the abundance of CODM and/or T6ODM transcripts indicated a direct physiological role for these enzymes in the metabolism of protopine alkaloids, and they revealed their indirect involvement in the formation of the antimicrobial benzo[c]phenanthridine sanguinarine and certain rhoeadine alkaloids in opium poppy.

  6. Neutrophils are Essential in Short Hairpin RNA of Indoleamine 2,3- Dioxygenase Mediated-antitumor Efficiency

    PubMed Central

    Liu, Kuan-Ting; Liu, Yao-Hua; Liu, Hsin-Liang; Chong, Inn-Wen; Yen, Meng-Chi; Kuo, Po-Lin

    2016-01-01

    Indoleamine 2,3-dioxygenase (IDO) is a rate limiting enzyme in tryptophan-degrading pathways and IDO activity results in immune suppression. Targeting IDO is a strategy of cancer immunotherapies. Our previous studies demonstrate that delivery of short hairpin against IDO (IDO shRNA) suppresses tumor growth and increases neutrophils infiltration into tumor. Neutrophils reveal antitumorigenic “N1” or protumorigenic “N2” phenotype in tumor microenvironment. However, the function of IDO shRNA-induced neutrophils is not clear. The LLC1 lung cancer model was used to investigate the role of these neutrophils. Intramuscular injection of IDO shRNA or IDO inhibitor treatment delayed tumor growth and both treatments increased neutrophil infiltration in tumor. Enriched tumor-infiltrating neutrophils expressed both high level of tumor necrosis factor-α and tumor necrosis factor-β (N1 and N2 associated molecules, respectively). In addition, IDO shRNA treatment induced interferon-γ and tryptophan transfer RNA expression in splenocytes. Systematic depletion of neutrophils abolished the IDO shRNA-induced therapeutic effect but did not affect the effect of IDO inhibitor. The levels of interferon-γ and tumor necrosis factor-α were suppressed in IDO shRNA treatment splenocytes after neutrophils depletion. In conclusion, these tumor-infiltrating neutrophils show antitumorigenic phenotype in spleen after IDO shRNA treatment in a murine lung cancer model. PMID:27922590

  7. Unity in diversity, a systems approach to regulating plant cell physiology by 2-oxoglutarate-dependent dioxygenases

    PubMed Central

    Kundu, Siddhartha

    2015-01-01

    Could a disjoint group of enzymes synchronize their activities and execute a complex multi-step, measurable, and reproducible response? Here, I surmise that the alpha-ketoglutarate dependent superfamily of non-haem iron (II) dioxygenases could influence cell physiology as a cohesive unit, and that the broad spectra of substrates transformed is an absolute necessity to this portrayal. This eclectic group comprises members from all major taxa, and participates in pesticide breakdown, hypoxia signaling, and osmotic stress neutralization. The oxidative decarboxylation of 2-oxoglutarate to succinate is coupled with a concomitant substrate hydroxylation and, in most cases, is followed by an additional specialized conversion. The domain profile of a protein sequence was used as an index of miscellaneous reaction chemistry and interpreted alongside existent kinetic data in a linear model of integrated function. Statistical parameters were inferred by the creation of a novel, empirically motivated flat-file database of over 3800 sequences (DB2OG) with putative 2-oxoglutarate dependent activity. The collated information was categorized on the basis of existing annotation schema. The data suggests that 2OG-dependent enzymes incorporate several desirable features of a systems level player. DB2OG, is free, accessible without a login to all users, and available at the following URL (http://comp-biol.theacms.in/DB2OG.html). PMID:25814993

  8. Ring-hydroxylating dioxygenase (RHD) expression in a microbial community during the early response to oil pollution.

    PubMed

    Paissé, Sandrine; Goñi-Urriza, Marisol; Stalder, Thibault; Stadler, Thibault; Budzinski, Hélène; Duran, Robert

    2012-04-01

    The early functional response of a bacterial community from the sediments of a chronically oil-polluted retention basin located at the Etang de Berre (France) was investigated just after petroleum addition. After removing hydrocarbon compounds by natural abiotic and biotic processes, the sediments were maintained in microcosms and Vic Bilh petroleum was added. The diversity and the expression of genes encoding ring-hydroxylating dioxygenases (RHD) were examined just after the petroleum addition until 14 days focussing on the first hours following the contamination. RHD gene copy numbers and diversity were maintained throughout all the incubation period; however, transcripts were detected only during the first 2 days. One dominant RHD gene, immediately and specifically expressed in response to petroleum contamination, was related to RHD gene carried by a plasmid found in Pseudomonas spp. The expression of the RHD genes was correlated with high biodegradation levels observed for low molecular weight PAHs at 7 days of incubation. The study shows that the bacterial metabolism induced just after the oil input is a key stage that could determine the bacterial community structure changes. Monitoring the expression of RHD genes, key genes involved in hydrocarbon degradation, may provide useful information for managing bioremediation processes.

  9. Dynamic changes in bacterial community structure and in naphthalene dioxygenase expression in vermicompost-amended PAH-contaminated soils.

    PubMed

    Di Gennaro, Patrizia; Moreno, Beatriz; Annoni, Emanuele; García-Rodríguez, Sonia; Bestetti, Giuseppina; Benitez, Emilio

    2009-12-30

    The aim of the present study was to explore the potential for using vermicompost from olive-mill waste as an organic amendment for enhanced bioremediation of polycyclic aromatic hydrocarbons (PAHs)-contaminated soils. The focus was to analyse the genetic potential and the naphthalene dioxygenase (NDO) expression of the bacterial communities involved in the degradation of naphthalene, as chemical model for the degradation of PAH. The structure of the metabolically active bacterial population was evidenced in the RNA-based denaturing gradient gel electrophoresis (DGGE) profiles. The relative expression of NDO was determined with real-time PCR in both the soil and the vermicompost cDNA. Naphthalene changed the structure of the metabolically active bacterial community in the vermicompost when this was artificially contaminated. When used as amendment, naphthalene-free vermicompost modified the bacterial population in the PAH-contaminated soil, evidenced in the DGGE gels after 1 month of incubation. In the amended soil, the vermicompost enhanced the NDO enzyme expression with a concomitant biodegradation of naphthalene. The effect of the vermicompost was to induce the expression of biodegradation indicator genes in the autochthonous bacterial community and/or incorporate new bacterial species capable of degrading PAH. The results indicated that vermicompost from olive-mill wastes could be considered a suitable technology to be used in PAH bioremediation.

  10. New Insight into the Cleavage Reaction of Nostoc sp. Strain PCC 7120 Carotenoid Cleavage Dioxygenase in Natural and Nonnatural Carotenoids

    PubMed Central

    Heo, Jinsol; Kim, Se Hyeuk

    2013-01-01

    Carotenoid cleavage dioxygenases (CCDs) are enzymes that catalyze the oxidative cleavage of carotenoids at a specific double bond to generate apocarotenoids. In this study, we investigated the activity and substrate preferences of NSC3, a CCD of Nostoc sp. strain PCC 7120, in vivo and in vitro using natural and nonnatural carotenoid structures. NSC3 cleaved β-apo-8′-carotenal at 3 positions, C-13C-14, C-15C-15′, and C-13′C-14′, revealing a unique cleavage pattern. NSC3 cleaves the natural structure of carotenoids 4,4′-diaponeurosporene, 4,4′-diaponeurosporen-4′-al, 4,4′-diaponeurosporen-4′-oic acid, 4,4′-diapotorulene, and 4,4′-diapotorulen-4′-al to generate novel cleavage products (apo-14′-diaponeurosporenal, apo-13′-diaponeurosporenal, apo-10′-diaponeurosporenal, apo-14′-diapotorulenal, and apo-10′-diapotorulenal, respectively). The study of carotenoids with natural or nonnatural structures produced by using synthetic modules could provide information valuable for understanding the cleavage reactions or substrate preferences of other CCDs in vivo and in vitro. PMID:23524669

  11. Absolute configuration-dependent epoxide formation from isoflavan-4-ol stereoisomers by biphenyl dioxygenase of Pseudomonas pseudoalcaligenes strain KF707.

    PubMed

    Seo, Jiyoung; Kang, Su-Il; Won, Dongho; Kim, Mihyang; Ryu, Ji-Young; Kang, Suk-Woo; Um, Byung-Hun; Pan, Cheol-Ho; Ahn, Joong-Hoon; Chong, Youhoon; Kanaly, Robert A; Han, Jaehong; Hur, Hor-Gil

    2011-03-01

    Biphenyl dioxygenase from Pseudomonas pseudoalcaligenes strain KF707 expressed in Escherichia coli was found to exhibit monooxygenase activity toward four stereoisomers of isoflavan-4-ol. LC-MS and LC-NMR analyses of the metabolites revealed that the corresponding epoxides formed between C2' and C3' on the B-ring of each isoflavan-4-ol substrate were the sole products. The relative reactivity of the stereoisomers was found to be in the order: (3S,4S)-cis-isoflavan-4-ol > (3R,4S)-trans-isoflavan-4-ol > (3S,4R)-trans-isoflavan-4-ol > (3R,4R)-cis-isoflavan-4-ol and this likely depended upon the absolute configuration of the 4-OH group on the isoflavanols, as explained by an enzyme-substrate docking study. The epoxides produced from isoflavan-4-ols by P. pseudoalcaligenes strain KF707 were further abiotically transformed into pterocarpan, the molecular structure of which is commonly found as part of plant-protective phytoalexins, such as maackiain from Cicer arietinum and medicarpin from Medicago sativa.

  12. A chromium(III)-superoxo complex in oxygen atom transfer reactions as a chemical model of cysteine dioxygenase.

    PubMed

    Cho, Jaeheung; Woo, Jaeyoung; Nam, Wonwoo

    2012-07-11

    Metal-superoxo species are believed to play key roles in oxygenation reactions by metalloenzymes. One example is cysteine dioxygenase (CDO) that catalyzes the oxidation of cysteine with O(2), and an iron(III)-superoxo species is proposed as an intermediate that effects the sulfoxidation reaction. We now report the first biomimetic example showing that a chromium(III)-superoxo complex bearing a macrocyclic TMC ligand, [Cr(III)(O(2))(TMC)(Cl)](+), is an active oxidant in oxygen atom transfer (OAT) reactions, such as the oxidation of phosphine and sulfides. The electrophilic character of the Cr(III)-superoxo complex is demonstrated unambiguously in the sulfoxidation of para-substituted thioanisoles. A Cr(IV)-oxo complex, [Cr(IV)(O)(TMC)(Cl)](+), formed in the OAT reactions by the chromium(III)-superoxo complex, is characterized by X-ray crystallography and various spectroscopic methods. The present results support the proposed oxidant and mechanism in CDO, such as an iron(III)-superoxo species is an active oxidant that attacks the sulfur atom of the cysteine ligand by the terminal oxygen atom of the superoxo group, followed by the formation of a sulfoxide and an iron(IV)-oxo species via an O-O bond cleavage.

  13. Altering substrate specificity of catechol 2,3-dioxygenase from Planococcus sp. strain S5 by random mutagenesis.

    PubMed

    Hupert-Kocurek, Katarzyna; Wojcieszyńska, Danuta; Guzik, Urszula

    2014-01-01

    c23o gene, encoding catechol 2,3-dioxygenase from Planococcus sp. strain S5 was randomly mutagenized to generate variant forms of the enzyme with higher degradation activity. Additionally, the effect of introduced mutations on the enzyme structure was analyzed based on the putative 3D models the wild-type and mutant enzymes. C23OB58 and C23OB81 mutant proteins with amino acid substitutions in close proximity to the enzyme surface or at the interface and in the vicinity of the enzyme active site respectively showed the lowest activity towards all catecholic substrates. The relative activity of C23OC61 mutant towards para-substituted catechols was 20-30% lower of the wild-type enzyme. In this mutant all changes: F191I, C268R, Y272H, V280A and Y293D were located within the conserved regions of C-terminal domain. From these F191I seems to have significant implications for enzyme activity. The highest activity towards different catechols was found for mutant C23OB65. R296Q mutation improved the activity of C23O especially against 4-chlorocatechol. The relative activity of above-mentioned mutant detected against this substrate was almost 6-fold higher than the wild-type enzyme. These results should facilitate future engineering of the enzyme for bioremediation.

  14. Refining the reaction mechanism of O2 towards its co-substrate in cofactor-free dioxygenases.

    PubMed

    Silva, Pedro J

    2016-01-01

    Cofactor-less oxygenases perform challenging catalytic reactions between singlet co-substrates and triplet oxygen, in spite of apparently violating the spin-conservation rule. In 1-H-3-hydroxy-4-oxoquinaldine-2,4-dioxygenase, the active site has been suggested by quantum chemical computations to fine tune triplet oxygen reactivity, allowing it to interact rapidly with its singlet substrate without the need for spin inversion, and in urate oxidase the reaction is thought to proceed through electron transfer from the deprotonated substrate to an aminoacid sidechain, which then feeds the electron to the oxygen molecule. In this work, we perform additional quantum chemical computations on these two systems to elucidate several intriguing features unaddressed by previous workers. These computations establish that in both enzymes the reaction proceeds through direct electron transfer from co-substrate to O2 followed by radical recombination, instead of minimum-energy crossing points between singlet and triplet potential energy surfaces without formal electron transfer. The active site does not affect the reactivity of oxygen directly but is crucial for the generation of the deprotonated form of the co-substrates, which have redox potentials far below those of their protonated forms and therefore may transfer electrons to oxygen without sizeable thermodynamic barriers. This mechanism seems to be shared by most cofactor-less oxidases studied so far.

  15. M30 Antagonizes Indoleamine 2,3-Dioxygenase Activation and Neurodegeneration Induced by Corticosterone in the Hippocampus.

    PubMed

    Lam, Chun-Sing; Tipoe, George Lim; Wong, Johnny Kong-Ching; Youdim, Moussa B H; Fung, Man-Lung

    2016-01-01

    Monoamine oxidases (MAO), downstream targets of glucocorticoid, maintain the turnover and homeostasis of monoamine neurotransmitters; yet, its pathophysiological role in monoamine deficiency, oxidative stress and neuroinflammation remains controversial. Protective effects of M30, a brain selective MAO inhibitor with iron-chelating antioxidant properties, have been shown in models of neurodegenerative diseases. This study aims to examine the neuroprotective mechanism of M30 against depressive-like behavior induced by corticosterone (CORT). Sprague-Dawley rats were given CORT subcutaneous injections with or without concomitant M30 administration for two weeks. CORT-treated rats exhibited depressive-like behavior with significant elevated levels of MAO activities, serotonin turnover, oxidative stress, neuroinflammation and apoptosis in the hippocampus with significant losses of synaptic proteins when compared to the control. The expression and activity of cytokine-responsive indoleamine 2,3-dioxygenase (IDO-1), a catabolic enzyme of serotonin and tryptophan, was significantly increased in the CORT-treated group with lowered levels of serotonin. Besides, CORT markedly reduced dendritic length and spine density. Remarkably, M30 administration neutralized the aberrant changes in the hippocampus and prevented the induction of depressive-like behavior induced by CORT. Our results suggest that M30 is neuroprotective against CORT-induced depression targeting elevated MAO activities that cause oxidative stress and neuroinflammation, resulting in IDO-1 activation, serotonin deficiency and neurodegeneration.

  16. M30 Antagonizes Indoleamine 2,3-Dioxygenase Activation and Neurodegeneration Induced by Corticosterone in the Hippocampus

    PubMed Central

    Lam, Chun-Sing; Tipoe, George Lim; Wong, Johnny Kong-Ching; Youdim, Moussa B. H.; Fung, Man-Lung

    2016-01-01

    Monoamine oxidases (MAO), downstream targets of glucocorticoid, maintain the turnover and homeostasis of monoamine neurotransmitters; yet, its pathophysiological role in monoamine deficiency, oxidative stress and neuroinflammation remains controversial. Protective effects of M30, a brain selective MAO inhibitor with iron-chelating antioxidant properties, have been shown in models of neurodegenerative diseases. This study aims to examine the neuroprotective mechanism of M30 against depressive-like behavior induced by corticosterone (CORT). Sprague-Dawley rats were given CORT subcutaneous injections with or without concomitant M30 administration for two weeks. CORT-treated rats exhibited depressive-like behavior with significant elevated levels of MAO activities, serotonin turnover, oxidative stress, neuroinflammation and apoptosis in the hippocampus with significant losses of synaptic proteins when compared to the control. The expression and activity of cytokine-responsive indoleamine 2,3-dioxygenase (IDO-1), a catabolic enzyme of serotonin and tryptophan, was significantly increased in the CORT-treated group with lowered levels of serotonin. Besides, CORT markedly reduced dendritic length and spine density. Remarkably, M30 administration neutralized the aberrant changes in the hippocampus and prevented the induction of depressive-like behavior induced by CORT. Our results suggest that M30 is neuroprotective against CORT-induced depression targeting elevated MAO activities that cause oxidative stress and neuroinflammation, resulting in IDO-1 activation, serotonin deficiency and neurodegeneration. PMID:27870896

  17. Theoretical approach to the innovative mutation of naphthalene 1,2-dioxygenase: a molecular dynamics and docking study.

    PubMed

    Librando, Vito; Pappalardo, Matteo

    2014-08-01

    Polycyclic aromatic hydrocarbons are a family of ubiquitous pollutants whose environmental behavior has been widely studied. Different bacterial species are able to decompose hydrocarbons by using them as a food source. One of the best-studied enzymes is naphthalene 1,2-dioxygenase (NDO). A practical way to optimize the degradation process is by mutating the protein involved, increasing both the degradation capacity of the enzyme and its ability to work under extreme environmental conditions of high temperature and low pH. Herein, we describe the study of NDO using molecular dynamics and docking calculations to discover new mutants with high degrading capabilities. We modeled eleven new mutants of NDO. The results indicate that increasing the size of the active site cavity in the mutants allowed for the insertion of high molecular weight PAHs. Additionally, the physicochemical properties of the NDO active sites make the sites well suited to interactions with PAHs, so most amino-acid modifications should not result in significantly altered behavior of NDO.

  18. Altering Catalytic Properties of 3-Chlorocatechol-Oxidizing Extradiol Dioxygenase from Sphingomonas xenophaga BN6 by Random Mutagenesis

    PubMed Central

    Riegert, Ulrich; Bürger, Sibylle; Stolz, Andreas

    2001-01-01

    The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism. PMID:11244073

  19. Altering catalytic properties of 3-chlorocatechol-oxidizing extradiol dioxygenase from Sphingomonas xenophaga BN6 by random mutagenesis.

    PubMed

    Riegert, U; Bürger, S; Stolz, A

    2001-04-01

    The 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas xenophaga strain BN6 (BphC1) oxidizes 3-chlorocatechol by a rather unique distal ring cleavage mechanism. In an effort to improve the efficiency of this reaction, bphC1 was randomly mutated by error-prone PCR. Mutants which showed increased activities for 3-chlorocatechol were obtained, and the mutant forms of the enzyme were shown to contain two or three amino acid substitutions. Variant enzymes containing single substitutions were constructed, and the amino acid substitutions responsible for altered enzyme properties were identified. One variant enzyme, which contained an exchanged amino acid in the C-terminal part, revealed a higher level of stability during conversion of 3-chlorocatechol than the wild-type enzyme. Two other variant enzymes contained amino acid substitutions in a region of the enzyme that is considered to be involved in substrate binding. These two variant enzymes exhibited a significantly altered substrate specificity and an about fivefold-higher reaction rate for 3-chlorocatechol conversion than the wild-type enzyme. Furthermore, these variant enzymes showed the novel capability to oxidize 3-methylcatechol and 2,3-dihydroxybiphenyl by a distal cleavage mechanism.

  20. Patchwork Assembly of nag-Like Nitroarene Dioxygenase Genes and the 3-Chlorocatechol Degradation Cluster for Evolution of the 2-Chloronitrobenzene Catabolism Pathway in Pseudomonas stutzeri ZWLR2-1▿

    PubMed Central

    Liu, Hong; Wang, Shu-Jun; Zhang, Jun-Jie; Dai, Hui; Tang, Huiru; Zhou, Ning-Yi

    2011-01-01

    Pseudomonas stutzeri ZWLR2-1 utilizes 2-chloronitrobenzene (2CNB) as a sole source of carbon, nitrogen, and energy. To identify genes involved in this pathway, a 16.2-kb DNA fragment containing putative 2CNB dioxygenase genes was cloned and sequenced. Of the products from the 19 open reading frames that resulted from this fragment, CnbAc and CnbAd exhibited striking identities to the respective α and β subunits of the Nag-like ring-hydroxylating dioxygenases involved in the metabolism of nitrotoluene, nitrobenzene, and naphthalene. The encoding genes were also flanked by two copies of insertion sequence IS6100. CnbAa and CnbAb are similar to the ferredoxin reductase and ferredoxin for anthranilate 1,2-dioxygenase from Burkholderia cepacia DBO1. Escherichia coli cells expressing cnbAaAbAcAd converted 2CNB to 3-chlorocatechol with concomitant nitrite release. Cell extracts of E. coli/pCNBC exhibited chlorocatechol 1,2-dioxygenase activity. The cnbCDEF gene cluster, homologous to a 3-chlorocatechol degradation cluster in Sphingomonas sp. strain TFD44, probably contains all of the genes necessary for the conversion of 3-chlorocatechol to 3-oxoadipate. The patchwork-like structure of this catabolic cluster suggests that the cnb cluster for 2CNB degradation evolved by recruiting two catabolic clusters encoding a nitroarene dioxygenase and a chlorocatechol degradation pathway. This provides another example to help elucidate the bacterial evolution of catabolic pathways in response to xenobiotic chemicals. PMID:21602392

  1. Purification and properties of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas putida mt-2 in comparison with that from Pseudomonas arvilla C-1.

    PubMed

    Nakai, C; Nakazawa, T; Nozaki, M

    1988-12-01

    Catechol 1,2-dioxygenase (pyrocatechase) has been purified to homogeneity from Pseudomonas putida mt-2. Most properties of this enzyme, such as the absorption spectrum, iron content, pH stability, pH optimum, substrate specificity, Km values, and amino acid composition, were similar to those of catechol 1,2-dioxygenase obtained from Pseudomonas arvilla C-1 [Y. Kojima et al. (1967) J. Biol. Chem. 242, 3270-3278]. These two catechol 1,2-dioxygenases were also found, from the results of Ouchterlony double diffusion, to share several antigenic determinants. The molecular weight of the putida enzyme was estimated to be 66,000 and 64,000 by sedimentation equilibrium analysis and Sephadex G-200 gel filtration, respectively. The enzyme gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, corresponding to Mr 32,000. The NH2-terminal sequence, which started with threonine, was determined up to 30 residues by Edman degradation. During the degradation, a single amino acid was released at each step. The NH2-terminal sequence up to 20 residues was identical to that of the beta subunit of the arvilla enzyme, with one exception at step 16, at which arginine was observed instead of glutamine. The COOH-terminal residue was deduced to be arginine on carboxypeptidase A and B digestions and on hydrazinolysis. These results indicate that the putida enzyme consists of two identical subunits, in contrast to the arvilla enzyme which consists of two nonidentical subunits, alpha and beta [C. Nakai et al. (1979) Arch. Biochem. Biophys. 195, 12-22], although these two enzymes have very similar properties.

  2. The Ternary Complex of PrnB (the Second Enzyme in the Pyrrolnitrin Biosynthesis Pathway), Tryptophan, and Cyanide Yields New Mechanistic Insights into the Indolamine Dioxygenase Superfamily*

    PubMed Central

    Zhu, Xiaofeng; van Pée, Karl-Heinz; Naismith, James H.

    2010-01-01

    Pyrrolnitrin (3-chloro-4-(2′-nitro-3′-chlorophenyl)pyrrole) is a broad-spectrum antifungal compound isolated from Pseudomonas pyrrocinia. Four enzymes (PrnA, PrnB, PrnC, and PrnD) are required for pyrrolnitrin biosynthesis from tryptophan. PrnB rearranges the indole ring of 7-Cl-l-tryptophan and eliminates the carboxylate group. PrnB shows robust activity in vivo, but in vitro activity for PrnB under defined conditions remains undetected. The structure of PrnB establishes that the enzyme belongs to the heme b-dependent indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) family. We report the cyanide complex of PrnB and two ternary complexes with both l-tryptophan or 7-Cl-l-tryptophan and cyanide. The latter two complexes are essentially identical and mimic the likely catalytic ternary complex that occurs during turnover. In the cyanide ternary complexes, a loop previously disordered becomes ordered, contributing to the binding of substrates. The conformations of the bound tryptophan substrates are changed from that seen previously in the binary complexes. In l-tryptophan ternary complex, the indole ring now adopts the same orientation as seen in the PrnB binary complexes with other tryptophan substrates. The amide and carboxylate group of the substrate are orientated in a new conformation. Tyr321 and Ser332 play a key role in binding these groups. The structures suggest that catalysis requires an l-configured substrate. Isothermal titration calorimetry data suggest d-tryptophan does not bind after cyanide (or oxygen) coordinates with the distal (or sixth) site of heme. This is the first ternary complex with a tryptophan substrate of a member of the tryptophan dioxygenase superfamily and has mechanistic implications. PMID:20421301

  3. Purification and some properties of 2-halobenzoate 1,2-dioxygenase, a two-component enzyme system from Pseudomonas cepacia 2CBS.

    PubMed Central

    Fetzner, S; Müller, R; Lingens, F

    1992-01-01

    The two components of the inducible 2-halobenzoate 1,2-dioxygenase from Pseudomonas cepacia 2CBS were purified to homogeneity. Yellow component B is a monomer (Mr, 37,500) with NADH-acceptor reductase activity. Ferricyanide, 2,6-dichlorophenol indophenol, and cytochrome c acted as electron acceptors. Component B was identified as an iron-sulfur flavoprotein containing 0.8 mol of flavin adenine dinucleotide, 1.7 mol of iron, and 1.7 mol of acid-labile sulfide per mol of enzyme. The isoelectric point was estimated to be pH 4.2. Component B was reduced by the addition of NADH. Red-brown component A (Mr, 200,000 to 220,000) is an iron-sulfur protein containing 5.8 mol of iron and 6.0 mol of acid-labile sulfide. The isoelectric point was within the range of pH 4.5 to 5.3. Component A could be reduced by dithionite or by NADH plus catalytic amounts of component B. Component A consisted of nonidentical subunits alpha (Mr, 52,000) and beta (Mr, 20,000). It contained approximately equimolar amounts of alpha and beta, and cross-linking studies suggested an alpha 3 beta 3 subunit structure of component A. The NADH- and Fe(2+)-dependent enzyme system was named 2-halobenzoate 1,2-dioxygenase, because it catalyzes the conversion of 2-fluoro-, 2-bromo-, 2-chloro-, and 2-iodobenzoate to catechol. 2-Halobenzoate 1,2-dioxygenase exhibited a very broad substrate specificity, but benzoate analogs with electron-withdrawing substituents at the ortho position were transformed preferentially. Images PMID:1370284

  4. Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system.

    PubMed Central

    Bünz, P V; Cook, A M

    1993-01-01

    Sphingomonas sp. strain RW1 synthesized a constitutive enzyme system that oxygenated dibenzofuran (DBF) to 2,2',3-trihydroxybiphenyl (THB). We purified this dibenzofuran 4,4a-dioxygenase system (DBFDOS) and found it to consist of four components which catalyzed three activities. Two isofunctional, monomeric flavoproteins (components A1 and A2; M(r) of about 44,000) transferred electrons from NADH to the second component (B; M(r) of about 12,000), a ferredoxin, which transported electrons to the heteromultimeric (alpha 2 beta 2) oxygenase component (C; M(r) of alpha, 45,000; M(r) of beta, 23,000). DBFDOS consumed 1 mol each of NADH, O2, and DBF, which was dioxygenated to about 1 mol of THB; no intermediate was observed. The reaction was thus the dioxygenation of DBF at the 4 and 4a positions to give a diene-diol-hemiacetal which rearomatized by spontaneous loss of a phenolate group to form THB. Components A1 and A2 each reduced dichlorophenolindophenol but had negligible activity with cytochrome c; each lost the yellow color, observed to be flavin adenine dinucleotide, upon purification. Component B, which transported electrons to the oxygenase or cytochrome c, had an N-terminal amino acid sequence with high homology to the putidaredoxin of cytochrome P-450cam. The oxygenase had the UV spectrum of a Rieske iron-sulfur center. We presume DBFDOS to be a class IIA dioxygenase system (EC 1.14.12.-), functionally similar to pyrazon dioxygenase. Images PMID:8407823

  5. Dibenzofuran 4,4a-dioxygenase from Sphingomonas sp. strain RW1: angular dioxygenation by a three-component enzyme system.

    PubMed

    Bünz, P V; Cook, A M

    1993-10-01

    Sphingomonas sp. strain RW1 synthesized a constitutive enzyme system that oxygenated dibenzofuran (DBF) to 2,2',3-trihydroxybiphenyl (THB). We purified this dibenzofuran 4,4a-dioxygenase system (DBFDOS) and found it to consist of four components which catalyzed three activities. Two isofunctional, monomeric flavoproteins (components A1 and A2; M(r) of about 44,000) transferred electrons from NADH to the second component (B; M(r) of about 12,000), a ferredoxin, which transported electrons to the heteromultimeric (alpha 2 beta 2) oxygenase component (C; M(r) of alpha, 45,000; M(r) of beta, 23,000). DBFDOS consumed 1 mol each of NADH, O2, and DBF, which was dioxygenated to about 1 mol of THB; no intermediate was observed. The reaction was thus the dioxygenation of DBF at the 4 and 4a positions to give a diene-diol-hemiacetal which rearomatized by spontaneous loss of a phenolate group to form THB. Components A1 and A2 each reduced dichlorophenolindophenol but had negligible activity with cytochrome c; each lost the yellow color, observed to be flavin adenine dinucleotide, upon purification. Component B, which transported electrons to the oxygenase or cytochrome c, had an N-terminal amino acid sequence with high homology to the putidaredoxin of cytochrome P-450cam. The oxygenase had the UV spectrum of a Rieske iron-sulfur center. We presume DBFDOS to be a class IIA dioxygenase system (EC 1.14.12.-), functionally similar to pyrazon dioxygenase.

  6. Structural insight into the expanded PCB-degrading abilities of a biphenyl dioxygenase obtained by directed evolution.

    PubMed

    Kumar, Pravindra; Mohammadi, Mahmood; Viger, Jean-François; Barriault, Diane; Gomez-Gil, Leticia; Eltis, Lindsay D; Bolin, Jeffrey T; Sylvestre, Michel

    2011-01-14

    The biphenyl dioxygenase of Burkholderia xenovorans LB400 is a multicomponent Rieske-type oxygenase that catalyzes the dihydroxylation of biphenyl and many polychlorinated biphenyls (PCBs). The structural bases for the substrate specificity of the enzyme's oxygenase component (BphAE(LB400)) are largely unknown. BphAE(p4), a variant previously obtained through directed evolution, transforms several chlorobiphenyls, including 2,6-dichlorobiphenyl, more efficiently than BphAE(LB400), yet differs from the parent oxygenase at only two positions: T335A/F336M. Here, we compare the structures of BphAE(LB400) and BphAE(p4) and examine the biochemical properties of two BphAE(LB400) variants with single substitutions, T335A or F336M. Our data show that residue 336 contacts the biphenyl and influences the regiospecificity of the reaction, but does not enhance the enzyme's reactivity toward 2,6-dichlorobiphenyl. By contrast, residue 335 does not contact biphenyl but contributes significantly to expansion of the enzyme's substrate range. Crystal structures indicate that Thr335 imposes constraints through hydrogen bonds and nonbonded contacts to the segment from Val320 to Gln322. These contacts are lost when Thr is replaced by Ala, relieving intramolecular constraints and allowing for significant movement of this segment during binding of 2,6-dichlorobiphenyl, which increases the space available to accommodate the doubly ortho-chlorinated congener 2,6-dichlorobiphenyl. This study provides important insight about how Rieske-type oxygenases can expand substrate range through mutations that increase the plasticity and/or mobility of protein segments lining the catalytic cavity.

  7. Purification and Characterization of Catechol 1,2-Dioxygenase from Acinetobacter sp. Y64 Strain and Escherichia coli Transformants.

    PubMed

    Lin, J; Milase, R N

    2015-12-01

    This study intends to purify and characterize catechol 1,2-dioxygenase (C1,2O) of phenol-degrading Acinetobacter sp. Y64 and of E. coli transformant. Acinetobacter sp. Y64 was capable of degrading 1000 mg/L of phenol within 14 ± 2 h at 30 °C, 160 rpm and pH of 7. One C1,2O of 36 kDa was purified using ammonium sulphate precipitation and Hitrap QFF column chromatograph with 49% recovery and a 10.6-fold increase in purity. Purified Y64 C1,2O had temperature and pH optimum at 37 °C and pH 7.7 respectively with the Michaelis constant of 17.53 µM and the maximal velocity of 1.95 U/mg, respectively. The presence of Fe(3+) or Fe(2+) enhanced the activity of Y64 C1,2O while other compounds such as Ca(2+), and EDTA had an inhibitory effect. 80% of C1,2O activity remained using 4-nitrocatechol as substrate while 2% remained using 3-methylcatechol compared with that using catechol. Y64 catA gene encoding C1,2O was amplified using PCR cloned into pET22b vector and expressed in Escherichia coli BL21 DE3 (pLysS) after transformation. Purified and cloned Y64 C1,2O show no significant differences in the biochemical properties. The phylogenetic tree based on the protein sequences indicates that these C1,2Os possess a common ancestry.

  8. Vascular Endothelial Expression of Indoleamine 2,3-Dioxygenase 1 Forms a Positive Gradient towards the Feto-Maternal Interface

    PubMed Central

    Blaschitz, Astrid; Gauster, Martin; Fuchs, Dietmar; Lang, Ingrid; Maschke, Petra; Ulrich, Daniela; Karpf, Eva; Takikawa, Osamu; Schimek, Michael G.; Dohr, Gottfried; Sedlmayr, Peter

    2011-01-01

    We describe the distribution of indoleamine 2,3-dioxygenase 1 (IDO1) in vascular endothelium of human first-trimester and term placenta. Expression of IDO1 protein on the fetal side of the interface extended from almost exclusively sub-trophoblastic capillaries in first-trimester placenta to a nearly general presence on villous vascular endothelia at term, including also most bigger vessels such as villous arteries and veins of stem villi and vessels of the chorionic plate. Umbilical cord vessels were generally negative for IDO1 protein. In the fetal part of the placenta positivity for IDO1 was restricted to vascular endothelium, which did not co-express HLA-DR. This finding paralleled detectability of IDO1 mRNA in first trimester and term tissue and a high increase in the kynurenine to tryptophan ratio in chorionic villous tissue from first trimester to term placenta. Endothelial cells isolated from the chorionic plate of term placenta expressed IDO1 mRNA in contrast to endothelial cells originating from human umbilical vein, iliac vein or aorta. In first trimester decidua we found endothelium of arteries rather than veins expressing IDO1, which was complementory to expression of HLA-DR. An estimation of IDO activity on the basis of the ratio of kynurenine and tryptophan in blood taken from vessels of the chorionic plate of term placenta indicated far higher values than those found in the peripheral blood of adults. Thus, a gradient of vascular endothelial IDO1 expression is present at both sides of the feto-maternal interface. PMID:21755000

  9. High activity of indoleamine 2,3 dioxygenase enzyme predicts disease severity and case fatality in bacteremic patients.

    PubMed

    Huttunen, Reetta; Syrjänen, Jaana; Aittoniemi, Janne; Oja, Simo S; Raitala, Annika; Laine, Janne; Pertovaara, Marja; Vuento, Risto; Huhtala, Heini; Hurme, Mikko

    2010-02-01

    Indoleamine 2,3-dioxygenase (IDO), which is the rate-limiting enzyme for tryptophan (trp) catabolism, may play a critical role in various inflammatory disorders. Recent studies on trauma patients have suggested that the degradation of trp is associated with the development of sepsis. The role of IDO activity in bacteremic patients is unclear. We studied IDO activity in 132 patients with bacteremia caused by Staphylococcus aureus, Streptococcus pneumoniae, beta-hemolytic streptococcae, or Eschericia coli. The serum concentrations of trp and its metabolite kynurenine (kyn) were measured by reverse-phase high-performance liquid chromatography 1 to 4 days after the positive blood culture and on recovery. The kyn-to-trp ratio (kyn/trp), reflecting the activity of the IDO enzyme, was calculated. The maximum value in the ratio for every patient during 1 to 4 days after positive blood culture was used in analysis. The maximum kyn/trp ratio was significantly higher in nonsurvivors versus those who survived (193.7 vs. 82.4 micromol/mmol; P = 0.001). The AUC(ROC) of maximal kyn/trp in the prediction of case fatality was 0.75 (95% confidence interval, 0.64-0.87), and the kyn/trp ratio at a cutoff level of 120 micromol/mmol showed 83% sensitivity and 69% specificity for fatal disease. A kyn/trp ratio greater than 120 micromol/mmol was associated with increased risk of death versus low (

  10. Structural Basis for Substrate and Oxygen Activation in Homoprotocatechuate 2,3-Dioxygenase: Roles of Conserved Active Site Histidine-200

    PubMed Central

    Kovaleva, Elena G.; Rogers, Melanie S.; Lipscomb, John D.

    2015-01-01

    Kinetic and spectroscopic studies have shown that the conserved active site residue His200 of the extradiol ring-cleaving homoprotocatechuate 2,3-dioxygenase (FeHPCD) from Brevibacterium fuscum is critical for efficient catalysis. The roles played by this residue are probed here by analysis of the steady state kinetics, pH dependence, and X-ray crystal structures of the FeHPCD position 200 variants His200Asn, His200Gln, and His200Glu alone and in complex with three catecholic substrates (homoprotocatechuate, 4-sulfonylcatechol, and 4-nitrocatechol) possessing substituents with different inductive capacity. Structures solved at 1.35 –1.75 Å resolution show that there is essentially no change in overall active site architecture or substrate binding mode for these variants when compared to the structures of the wild type enzyme and its analogous complexes. This shows that the maximal 50-fold decrease in kcat for ring cleavage, the dramatic changes in pH dependence, and the switch from ring cleavage to ring oxidation of 4-nitrocatechol by the FeHPCD variants can be attributed specifically to the properties of the altered second sphere residue and the substrate. The results suggest that proton transfer is necessary for catalysis, and that it occurs most efficiently when the substrate provides the proton and His200 serves as a catalyst. However, in the absence of an available substrate proton, a defined proton-transfer pathway in the protein can be utilized. Changes in steric bulk and charge of the residue at position 200 appear capable of altering the rate-limiting step in catalysis, and perhaps, the nature of the reactive species. PMID:26267790

  11. Attenuation of high sucrose diet–induced insulin resistance in tryptophan 2,3-dioxygenase deficient Drosophila melanogaster vermilion mutants

    PubMed Central

    Navrotskaya, Valeriya; Oxenkrug, Gregory; Vorobyova, Lyudmila; Summergrad, Paul

    2015-01-01

    Exposure to high sugar diet (HSD) serves as an experimental model of insulin resistance (IR) and type 2 diabetes (T2D) in mammals and insects. Peripheral IR induced by HSD delays emergence of pupae from larvae and decreases body weight of Drosophila imago. Understanding of mechanisms of IR/T2D is essential for refining T2D prevention and treatment strategies. Dysregulation of tryptophan (TRP) – kynurenine (KYN) pathway was suggested as one of the mechanisms of IR development. Rate-limiting enzyme of TRP – KYN pathway in Drosophila is TRP 2,3-dioxygenase (TDO), an evolutionary conserved ortholog of human TDO. In insects TDO is encoded by vermilion gene. TDO is not active in vermilion mutants. In order to evaluate the possible impact of deficient formation of KYN from TRP on the inducement of IR by HSD, we compared the effect of HSD in wild type (Oregon) and vermilion mutants of Drosophila melanogaster by assessing the time of white pupae emergence from larva and body weight of imago. Delay of emergence of pupae from larvae induced by high sucrose diet was less pronounced in vermilion (1.4 days) than in Oregon flies (3.3 days) in comparison with flies maintained on standard diet. Exposure to high sucrose diet decreased body weight of Oregon (but not vermilion) imago. Attenuation of high sucrose diet–induced IR/T2D in vermilion flies might depend on deficiency of TRP – KYN pathway. Besides IR/T2D, HSD induces obesity in Drosophila. Future studies of HSD-induced obesity and IR/T2D in TDO deficient vermilion mutants of Drosophila might help to understand the mechanisms of high association between IR/T2D and obesity. Modulation of TRP – KYN metabolism might be utilized for prevention and treatment of IR/T2D. PMID:26191458

  12. Reaction coordinate analysis for beta-diketone cleavage by the non-heme Fe2+-dependent dioxygenase Dke1.

    PubMed

    Straganz, Grit D; Nidetzky, Bernd

    2005-09-07

    Acetylacetone dioxygenase from Acinetobacter johnsonii (Dke1) utilizes a non-heme Fe2+ cofactor to promote dioxygen-dependent conversion of 2,4-pentanedione (PD) into methylglyoxal and acetate. An oxidative carbon-carbon bond cleavage by Dke1 is triggered from a C-3 peroxidate intermediate that performs an intramolecular nucleophilic attack on the adjacent carbonyl group. But how does Dke1 bring about the initial reduction of dioxygen? To answer this question, we report here a reaction coordinate analysis for the part of the Dke1 catalytic cycle that involves O2 chemistry. A weak visible absorption band (epsilon approximately 0.2 mM(-1) cm(-1)) that is characteristic of an enzyme-bound Fe2+-beta-keto-enolate complex served as spectroscopic probe of substrate binding and internal catalytic steps. Transient and steady-state kinetic studies reveal that O2-dependent conversion of the chromogenic binary complex is rate-limiting for the overall reaction. Linear free-energy relationship analysis, in which apparent turnover numbers (k(app) cat) for enzymatic bond cleavage of a series of substituted beta-dicarbonyl substrates were correlated with calculated energies for the highest occupied molecular orbitals of the corresponding beta-keto-enolate structures, demonstrates unambiguously that k(app) cat is governed by the electron-donating ability of the substrate. The case of 2'-hydroxyacetophenone (2'HAP), a completely inactive beta-dicarbonyl analogue that has the enol double bond delocalized into the aromatic ring, indicates that dioxygen reduction and C-O bond formation cannot be decoupled and therefore take place in one single kinetic step.

  13. The cystine/glutamate antiporter regulates indoleamine 2,3-dioxygenase protein levels and enzymatic activity in human dendritic cells.

    PubMed

    Mattox, Mildred L; D'Angelo, June A; Grimes, Zachary M; Fiebiger, Edda; Dickinson, Bonny L

    2012-11-30

    Indoleamine 2,3-dioxygenase (IDO) is the rate-limiting enzyme in the tryptophan-catabolizing pathway and a key regulator of peripheral immune tolerance. As the suppressive effects of IDO are predominantly mediated by dendritic cells (DCs) and IDO-competent DCs promote long-term immunologic tolerance, a detailed understanding of how IDO expression and activity is regulated in these cells is central to the rational design of therapies to induce robust immune tolerance. We previously reported that the cystine/glutamate antiporter modulates the functional expression of IDO in human monocyte-derived DCs. Specifically, we showed that blocking antiporter uptake of cystine significantly increased both IDO mRNA and IDO enzymatic activity and that this correlated with impaired DC presentation of exogenous antigen to T cells via MHC class II and the cross-presentation pathway. The antiporter regulates intracellular and extracellular redox by transporting cystine into the cell in exchange for glutamate. Intracellular cystine is reduced to cysteine to support biosynthesis of the major cellular antioxidant glutathione and cysteine is exported from the cell where it functions as an extracellular antioxidant. Here we show that antiporter control of IDO expression in DCs is reversible, independent of interferon-γ, regulated by redox, and requires active protein synthesis. These findings highlight a role for antiporter regulation of cellular redox as a critical control point for modulating IDO expression and activity in DCs. Thus, systemic disease and aging, processes that perturb redox homeostasis, may adversely affect immunity by promoting the generation of IDO-competent DCs.

  14. Respiratory Syncytial Virus-Infected Mesenchymal Stem Cells Regulate Immunity via Interferon Beta and Indoleamine-2,3-Dioxygenase

    PubMed Central

    Cheung, Michael B.; Sampayo-Escobar, Viviana; Green, Ryan; Moore, Martin L.; Mohapatra, Subhra; Mohapatra, Shyam S.

    2016-01-01

    Respiratory syncytial virus (RSV) has been reported to infect human mesenchymal stem cells (MSCs) but the consequences are poorly understood. MSCs are present in nearly every organ including the nasal mucosa and the lung and play a role in regulating immune responses and mediating tissue repair. We sought to determine whether RSV infection of MSCs enhances their immune regulatory functions and contributes to RSV-associated lung disease. RSV was shown to replicate in human MSCs by fluorescence microscopy, plaque assay, and expression of RSV transcripts. RSV-infected MSCs showed differentially altered expression of cytokines and chemokines such as IL-1β, IL6, IL-8 and SDF-1 compared to epithelial cells. Notably, RSV-infected MSCs exhibited significantly increased expression of IFN-β (~100-fold) and indoleamine-2,3-dioxygenase (IDO) (~70-fold) than in mock-infected MSCs. IDO was identified in cytosolic protein of infected cells by Western blots and enzymatic activity was detected by tryptophan catabolism assay. Treatment of PBMCs with culture supernatants from RSV-infected MSCs reduced their proliferation in a dose dependent manner. This effect on PBMC activation was reversed by treatment of MSCs with the IDO inhibitors 1-methyltryptophan and vitamin K3 during RSV infection, a result we confirmed by CRISPR/Cas9-mediated knockout of IDO in MSCs. Neutralizing IFN-β prevented IDO expression and activity. Treatment of MSCs with an endosomal TLR inhibitor, as well as a specific inhibitor of the TLR3/dsRNA complex, prevented IFN-β and IDO expression. Together, these results suggest that RSV infection of MSCs alters their immune regulatory function by upregulating IFN-β and IDO, affecting immune cell proliferation, which may account for the lack of protective RSV immunity and for chronicity of RSV-associated lung diseases such as asthma and COPD. PMID:27695127

  15. A novel l-isoleucine-4′-dioxygenase and l-isoleucine dihydroxylation cascade in Pantoea ananatis

    PubMed Central

    Smirnov, Sergey V; Sokolov, Pavel M; Kotlyarova, Veronika A; Samsonova, Natalya N; Kodera, Tomohiro; Sugiyama, Masakazu; Torii, Takayoshi; Hibi, Makoto; Shimizu, Sakayu; Yokozeki, Kenzo; Ogawa, Jun

    2013-01-01

    A unique operon structure has been identified in the genomes of several plant- and insect-associated bacteria. The distinguishing feature of this operon is the presence of tandem hilA and hilB genes encoding dioxygenases belonging to the PF13640 and PF10014 (BsmA) Pfam families, respectively. The genes encoding HilA and HilB from Pantoea ananatis AJ13355 were cloned and expressed in Escherichia coli. The culturing of E. coli cells expressing hilA (E. coli-HilA) or both hilA and hilB (E. coli-HilAB) in the presence of l-isoleucine resulted in the conversion of l-isoleucine into two novel biogenic compounds: l-4′-isoleucine and l-4,4′-dihydroxyisoleucine, respectively. In parallel, two novel enzymatic activities were detected in the crude cell lysates of the E. coli-HilA and E. coli-HilAB strains: l-isoleucine, 2-oxoglutarate: oxygen oxidoreductase (4′-hydroxylating) (HilA) and l-4′-hydroxyisoleucine, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating) (HilB), respectively. Two hypotheses regarding the physiological significance of C-4(4′)-hydroxylation of l-isoleucine in bacteria are also discussed. According to first hypothesis, the l-isoleucine dihydroxylation cascade is involved in synthesis of dipeptide antibiotic in P. ananatis. Another unifying hypothesis is that the C-4(4′)-hydroxylation of l-isoleucine in bacteria could result in the synthesis of signal molecules belonging to two classes: 2(5H)-furanones and analogs of N-acyl homoserine lactone. PMID:23554367

  16. Common genetic variation in the indoleamine-2,3-dioxygenase genes and antidepressant treatment outcome in major depressive disorder.

    PubMed

    Cutler, Jessica A; Rush, A John; McMahon, Francis J; Laje, Gonzalo

    2012-03-01

    The essential amino acid tryptophan is the precursor to serotonin, but it can also be metabolized into kynurenine through indoleamine-2,3-dioxygenase (IDO). Increased immune activation has long been associated with symptoms of depression and has been shown to upregulate the expression of IDO. The presence of additional IDO directs more tryptophan down the kynurenine pathway, leaving less available for synthesis of serotonin and its metabolites. Kynurenine can be metabolized through a series of enzymes to quinolinic acid, a potent N-methyl-D-aspartate receptor agonist with demonstrated neurotoxic effects. We tested the hypothesis that IDO plays a role in outcome of treatment with the selective serotonin reuptake inhibitor, citalopram. Patients consisted of 1953 participants enrolled in the Sequenced Treatment Alternatives to Relieve Depression study (STAR*D). Genotypes corresponding to 94 single nucleotide polymorphisms (SNPs) in the genes IDO1 and IDO2, which encode IDO and IDO2, were extracted from a larger genome-wide set and analyzed using single marker tests to look for association with previously defined response, remission and QIDS-C score change phenotypes, with adequate correction for racial stratification and multiple testing. One SNP, rs2929115, showed evidence of association with citalopram response (OR = 0.64, p = 0.0005) after experiment-wide correction for multiple testing. Another closely associated marker, rs2929116 (OR = 0.64, p = 0.0006) had an experiment-wide significant result. Both implicated SNPs are located between 26 kb and 28 kb downstream of IDO2. We conclude that common genetic variation in IDO1 and IDO2 may play a role in antidepressant treatment outcome. These results are modest in a genome-wide context and need to be replicated in an independent sample.

  17. Involvement of the Cys-Tyr cofactor on iron binding in the active site of human cysteine dioxygenase.

    PubMed

    Arjune, Sita; Schwarz, Guenter; Belaidi, Abdel A

    2015-01-01

    Sulfur metabolism has gained increasing medical interest over the last years. In particular, cysteine dioxygenase (CDO) has been recognized as a potential marker in oncology due to its altered gene expression in various cancer types. Human CDO is a non-heme iron-dependent enzyme, which catalyzes the irreversible oxidation of cysteine to cysteine sulfinic acid, which is further metabolized to taurine or pyruvate and sulfate. Several studies have reported a unique post-translational modification of human CDO consisting of a cross-link between cysteine 93 and tyrosine 157 (Cys-Tyr), which increases catalytic efficiency in a substrate-dependent manner. However, the reaction mechanism by which the Cys-Tyr cofactor increases catalytic efficiency remains unclear. In this study, steady-state kinetics were determined for wild type CDO and two different variants being either impaired or saturated with the Cys-Tyr cofactor. Cofactor formation in CDO resulted in an approximately fivefold increase in k cat and tenfold increase in k cat/K m over the cofactor-free CDO variant. Furthermore, iron titration experiments revealed an 18-fold decrease in K d of iron upon cross-link formation. This finding suggests a structural role of the Cys-Tyr cofactor in coordinating the ferrous iron in the active site of CDO in accordance with the previously postulated reaction mechanism of human CDO. Finally, we identified product-based inhibition and α-ketoglutarate and glutarate as CDO inhibitors using a simplified well plate-based activity assay. This assay can be used for high-throughput identification of additional inhibitors, which may contribute to understand the functional importance of CDO in sulfur amino acid metabolism and related diseases.

  18. High expression of IMPACT protein promotes resistance to indoleamine 2,3-dioxygenase-induced cell death.

    PubMed

    Habibi, Darya; Jalili, Reza B; Forouzandeh, Farshad; Ong, Christopher J; Ghahary, Aziz

    2010-10-01

    Indoleamine 2,3-dioxygenase (IDO), a tryptophan degrading enzyme, is a potent immunomodulatory factor. IDO expression in fibroblasts selectively induces apoptosis in immune cells but not in primary skin cells. However, the mechanism(s) of this selective effect of IDO-induced low tryptophan environment is not elucidated. The aim of present study was to investigate whether the activity of general control non-derepressible-2(GCN2) kinase stress-responsive pathway and its known inhibitor, protein IMPACT homolog, in immune and skin cells are differentially regulated in response to IDO-induced low tryptophan environment. IDO-expressing human fibroblasts were co-cultured with Jurkat cells, human T cells, fibroblasts, or keratinocytes. Activation of GCN2 pathway was significantly higher in immune cells exposed to IDO-expressing environment relative to that of skin cells. In contrast, IMPACT was highly and constitutively expressed in skin cells while its expression was very low in stimulated T cells and undetectable in Jurkat cells. A significant IDO-induced suppressive as well as apoptotic effect was demonstrated in IMPACT knocked down fibroblasts co-cultured with IDO-expressing fibroblasts. Proliferation of Jurkat cells, stably transduced with IMPACT-expressing vector, was rescued significantly in tryptophan-deficient but not IDO-expressing environment. This may be due to the ability of IMPACT to recover the effects of IDO-mediated tryptophan depletion (GCN2 dependent) but not the effects of IDO-generated cytotoxic metabolites. These findings collectively suggest for the first time that high expression of protein IMPACT homolog in non-immune cells such as skin cells acts as a protective mechanism against IDO-induced GCN2 activation, therefore, makes them resistant to the amino acid-deprived environment caused by IDO.

  19. Suppression of Immunodominant Antitumor and Antiviral CD8+ T Cell Responses by Indoleamine 2,3-Dioxygenase

    PubMed Central

    Atef Yekta, Maryam; Szabo, Peter A.; Garg, Nitan; Schell, Todd D.; Jevnikar, Anthony M.; Sharif, Shayan; Singh, Bhagirath; Haeryfar, S. M. Mansour

    2014-01-01

    Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme known to suppress antitumor CD8+ T cells (TCD8). The role of IDO in regulation of antiviral TCD8 responses is far less clear. In addition, whether IDO controls both immunodominant and subdominant TCD8 is not fully understood. This is an important question because the dominance status of tumor- and virus-specific TCD8 may determine their significance in protective immunity and in vaccine design. We evaluated the magnitude and breadth of cross-primed TCD8 responses to simian virus 40 (SV40) large T antigen as well as primary and recall TCD8 responses to influenza A virus (IAV) in the absence or presence of IDO. IDO−/− mice and wild-type mice treated with 1-methyl-D-tryptophan, a pharmacological inhibitor of IDO, exhibited augmented responses to immunodominant epitopes encoded by T antigen and IAV. IDO-mediated suppression of these responses was independent of CD4+CD25+FoxP3+ regulatory T cells, which remained numerically and functionally intact in IDO−/− mice. Treatment with L-kynurenine failed to inhibit TCD8 responses, indicating that tryptophan metabolites are not responsible for the suppressive effect of IDO in our models. Immunodominant T antigen-specific TCD8 from IDO−/− mice showed increased Ki-67 expression, suggesting that they may have acquired a more vigorous proliferative capacity in vivo. In conclusion, IDO suppresses immunodominant TCD8 responses to tumor and viral antigens. Our work also demonstrates that systemic primary and recall TCD8 responses to IAV are controlled by IDO. Inhibition of IDO thus represents an attractive adjuvant strategy in boosting anticancer and antiviral TCD8 targeting highly immunogenic antigens. PMID:24587363

  20. Structural Study of a Flexible Active Site Loop in Human Indoleamine 2,3-Dioxygenase and Its Functional Implications.

    PubMed

    Álvarez, Lucía; Lewis-Ballester, Ariel; Roitberg, Adrián; Estrin, Darío A; Yeh, Syun-Ru; Marti, Marcelo A; Capece, Luciana

    2016-05-17

    Human indoleamine 2,3-dioxygenase catalyzes the oxidative cleavage of tryptophan to N-formyl kynurenine, the initial and rate-limiting step in the kynurenine pathway. Additionally, this enzyme has been identified as a possible target for cancer therapy. A 20-amino acid protein segment (the JK loop), which connects the J and K helices, was not resolved in the reported hIDO crystal structure. Previous studies have shown that this loop undergoes structural rearrangement upon substrate binding. In this work, we apply a combination of replica exchange molecular dynamics simulations and site-directed mutagenesis experiments to characterize the structure and dynamics of this protein region. Our simulations show that the JK loop can be divided into two regions: the first region (JK loop(C)) displays specific and well-defined conformations and is within hydrogen bonding distance of the substrate, while the second region (JK loop(N)) is highly disordered and exposed to the solvent. The peculiar flexible nature of JK loop(N) suggests that it may function as a target for post-translational modifications and/or a mediator for protein-protein interactions. In contrast, hydrogen bonding interactions are observed between the substrate and Thr379 in the highly conserved "GTGG" motif of JK loop(C), thereby anchoring JK loop(C) in a closed conformation, which secures the appropriate substrate binding mode for catalysis. Site-directed mutagenesis experiments confirm the key role of this residue, highlighting the importance of the JK loop(C) conformation in regulating the enzymatic activity. Furthermore, the existence of the partially and totally open conformations in the substrate-free form suggests a role of JK loop(C) in controlling substrate and product dynamics.

  1. Molecular cloning and characterization of cDNAs encoding carotenoid cleavage dioxygenase in bitter melon (Momordica charantia).

    PubMed

    Tuan, Pham Anh; Park, Sang Un

    2013-01-01

    Carotenoid cleavage dioxygenases (CCDs) are a family of enzymes that catalyze the oxidative cleavage of carotenoids at various chain positions to form a broad spectrum of apocarotenoids, including aromatic substances, pigments and phytohormones. Using the rapid amplification of cDNA ends (RACE) PCR method, we isolated three cDNA-encoding CCDs (McCCD1, McCCD4, and McNCED) from Momordica charantia. Amino acid sequence alignments showed that they share high sequence identity with other orthologous genes. Quantitative real-time RT PCR (reverse transcriptase PCR) analysis revealed that the expression of McCCD1 and McCCD4 was highest in flowers, and lowest in roots and old leaves (O-leaves). During fruit maturation, the two genes displayed differential expression, with McCCD1 peaking at mid-stage maturation while McCCD4 showed the lowest expression at that stage. The mRNA expression level of McNCED, a key enzyme involved in abscisic acid (ABA) biosynthesis, was high during fruit maturation and further increased at the beginning of seed germination. When first-leaf stage plants of M. charantia were exposed to dehydration stress, McNCED mRNA expression was induced primarily in the leaves and, to a lesser extend, in roots and stems. McNCED expression was also induced by high temperature and salinity, while treatment with exogenous ABA led to a decrease. These results should be helpful in determining the substrates and cleavage sites catalyzed by CCD genes in M. charantia, and also in defining the roles of CCDs in growth and development, and in the plant's response to environmental stress.

  2. Synergistic antitumor effect with indoleamine 2,3-dioxygenase inhibition and temozolomide in a murine glioma model.

    PubMed

    Hanihara, Mitsuto; Kawataki, Tomoyuki; Oh-Oka, Kyoko; Mitsuka, Kentaro; Nakao, Atsuhito; Kinouchi, Hiroyuki

    2016-06-01

    OBJECT Indoleamine 2,3-dioxygenase (IDO), a key enzyme of tryptophan (Trp) metabolism, is involved in tumor-derived immune suppression through depletion of Trp and accumulation of the metabolite kynurenine, resulting in inactivation of natural killer cells and generation of regulatory T cells (Tregs). It has been reported that high expression of IDO in cancer cells is associated with suppression of the antitumor immune response and is consistent with a poor prognosis. Thus, IDO may be a therapeutic target for malignant cancer. The authors have recently shown that IDO expression is markedly increased in human glioblastoma and secondary glioblastoma with malignant change, suggesting that IDO targeting may also have therapeutic potential for patients with glioma. The aim of this study was to investigate the antitumor effect of IDO inhibition and to examine the synergistic function of IDO inhibitor and temozolomide (TMZ) in a murine glioma model. METHODS Murine glioma GL261 cells and human glioma U87 cells were included in this study. The authors used 3 mouse models to study glioma cell growth: 1) a subcutaneous ectopic model, 2) a syngeneic intracranial orthotopic model, and 3) an allogenic intracranial orthotopic model. IDO inhibition was achieved via knockdown of IDO in GL261 cells using short hairpin RNA (shRNA) and through oral administration of the IDO inhibitor, 1-methyl-l-tryptophan (1-MT). Tumor volume in the subcutaneous model and survival time in the intracranial model were evaluated. RESULTS In the subcutaneous model, oral administration of 1-MT significantly suppressed tumor growth, and synergistic antitumor effects of 1-MT and TMZ were observed (p < 0.01). Mice containing intracranially inoculated IDO knockdown cells had a significantly longer survival period as compared with control mice (p < 0.01). CONCLUSIONS These results suggest that IDO expression is implicated in immunosuppression and tumor progression in glioma cells. Therefore, combining IDO

  3. Inhibition of indoleamine 2,3-dioxygenase 1 expression alters immune response in colon tumor microenvironment in mice

    PubMed Central

    Takamatsu, Manabu; Hirata, Akihiro; Ohtaki, Hirofumi; Hoshi, Masato; Ando, Tatsuya; Ito, Hiroyasu; Hatano, Yuichiro; Tomita, Hiroyuki; Kuno, Toshiya; Saito, Kuniaki; Seishima, Mitsuru; Hara, Akira

    2015-01-01

    Indoleamine 2,3-dioxygenase (IDO), an enzyme that degrades the essential amino acid l-tryptophan along the kynurenine pathway, exerts immunomodulatory effects in a number of diseases. IDO expression is increased in tumor tissue and in draining lymph nodes; this increase is thought to play a role in tumor evasion by suppressing the immune response. A competitive inhibitor of IDO is currently being tested in clinical trials for the treatment of relapsed or refractory solid tumors, but the efficacy of IDO inhibition in colorectal tumors remains to be fully elucidated. In this study, we investigated the effect of IDO deficiency on colon tumorigenesis in mice by genetic deletion and pharmacological inhibition. Ido1-deficient(−/−) mice were crossed with ApcMin/+ mice or were administered azoxymethane with or without dextran sodium sulfate. Ido1 deficiency did not lead to significant differences in the size and number of colon tumors. Similarly, the pharmacological inhibition of IDO using 1-methyltryptophan (1-mT) also resulted in no significant differences in tumor size and number in ApcMin/+ mice. However, Ido1 deficiency altered the immune response in the tumor microenvironment, showing a significant increase in mRNA expression of pro-inflammatory cytokines and a significant decrease in the number of Foxp3-positive regulatory T cells in the colon tumors of Ido1(−/−) mice. Importantly, 1-mT treatment also significantly altered cytokine expression in the colon tumor tissues. These results suggest that IDO inhibition alone cannot sufficiently suppress colon cancer development in mice despite its immunomodulatory activity in the tumor microenvironment. PMID:26033215

  4. Induction of Indoleamine 2, 3-Dioxygenase in Human Dendritic Cells by a Cholera Toxin B Subunit—Proinsulin Vaccine

    PubMed Central

    Mbongue, Jacques C.; Nicholas, Dequina A.; Zhang, Kangling; Kim, Nan-Sun; Hamilton, Brittany N.; Larios, Marco; Zhang, Guangyu; Umezawa, Kazuo; Firek, Anthony F.; Langridge, William H. R.

    2015-01-01

    Dendritic cells (DC) interact with naïve T cells to regulate the delicate balance between immunity and tolerance required to maintain immunological homeostasis. In this study, immature human dendritic cells (iDC) were inoculated with a chimeric fusion protein vaccine containing the pancreatic β-cell auto-antigen proinsulin linked to a mucosal adjuvant the cholera toxin B subunit (CTB-INS). Proteomic analysis of vaccine inoculated DCs revealed strong up-regulation of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1). Increased biosynthesis of the immunosuppressive enzyme was detected in DCs inoculated with the CTB-INS fusion protein but not in DCs inoculated with proinsulin, CTB, or an unlinked combination of the two proteins. Immunoblot and PCR analyses of vaccine treated DCs detected IDO1mRNA by 3 hours and IDO1 protein synthesis by 6 hours after vaccine inoculation. Determination of IDO1 activity in vaccinated DCs by measurement of tryptophan degradation products (kynurenines) showed increased tryptophan cleavage into N-formyl kynurenine. Vaccination did not interfere with monocytes differentiation into DC, suggesting the vaccine can function safely in the human immune system. Treatment of vaccinated DCs with pharmacological NF-κB inhibitors ACHP or DHMEQ significantly inhibited IDO1 biosynthesis, suggesting a role for NF-κB signaling in vaccine up-regulation of dendritic cell IDO1. Heat map analysis of the proteomic data revealed an overall down-regulation of vaccinated DC functions, suggesting vaccine suppression of DC maturation. Together, our experimental data indicate that CTB-INS vaccine induction of IDO1 biosynthesis in human DCs may result in the inhibition of DC maturation generating a durable state of immunological tolerance. Understanding how CTB-INS modulates IDO1 activity in human DCs will facilitate vaccine efficacy and safety, moving this immunosuppressive strategy closer to clinical applications for prevention of type 1

  5. Structure of Naegleria Tet-like dioxygenase (NgTet1) in complexes with a reaction intermediate 5-hydroxymethylcytosine DNA

    DOE PAGES

    Hashimoto, Hideharu; Pais, June E.; Dai, Nan; ...

    2015-08-31

    The family of ten-eleven translocation (Tet) dioxygenases is widely distributed across the eukaryotic tree of life, from mammals to the amoeboflagellate Naegleria gruberi. Like mammalian Tet proteins, the Naegleria Tet-like protein, NgTet1, acts on 5-methylcytosine (5mC) and generates 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) in three consecutive, Fe(II)- and α-ketoglutarate-dependent oxidation reactions. The two intermediates, 5hmC and 5fC, could be considered either as the reaction product of the previous enzymatic cycle or the substrate for the next cycle. Here we present a new crystal structure of NgTet1 in complex with DNA containing a 5hmC. Along with the previously solvedmore » NgTet1–5mC structure, the two complexes offer a detailed picture of the active site at individual stages of the reaction cycle. In the crystal, the hydroxymethyl (OH-CH2-) moiety of 5hmC points to the metal center, representing the reaction product of 5mC hydroxylation. The hydroxyl oxygen atom could be rotated away from the metal center, to a hydrophobic pocket formed by Ala212, Val293 and Phe295. Such rotation turns the hydroxyl oxygen atom away from the product conformation, and exposes the target CH2 towards the metal-ligand water molecule, where a dioxygen O2 molecule would occupy to initiate the next round of reaction by abstracting a hydrogen atom from the substrate. The Ala212-to-Val (A212V) mutant profoundly limits the product to 5hmC, probably due to the reduced hydrophobic pocket size restricts the binding of 5hmC as a substrate.« less

  6. Elevated interleukin-27 levels in human neonatal macrophages regulate indoleamine dioxygenase in a STAT-1 and STAT-3-dependent manner.

    PubMed

    Jung, Joo-Yong; Gleave Parson, Madeline; Kraft, Jennifer D; Lyda, Logan; Kobe, Brianna; Davis, Celestia; Robinson, Jembber; Peña, Maria Marjorette O; Robinson, Cory M

    2016-09-01

    Microbial infections are a major cause of infant mortality as a result of limitations in immune defences. Interleukin-27 (IL-27) is a heterodimeric cytokine produced primarily by leucocytes and is immunosuppressive toward lymphocytes and leucocytes. Our laboratory demonstrated that human neonatal macrophages express IL-27 more abundantly than adult macrophages. Similarly in mice, IL-27 expression is elevated early in life and maintained through infancy. To determine IL-27-regulated mechanisms that may limit immunity, we evaluated the expression of a number of genes in response to this cytokine in primary human neonatal macrophages. Indoleamine 2,3-dioxygenase (IDO) gene expression was increased dose-responsively by IL-27. We have previously demonstrated inhibition of T-cell proliferation and cytokine production by neonatal macrophage-generated IL-27, and IDO is often implicated in this negative regulation. An increase in IDO protein was demonstrated by immunofluorescence microscopy and was consistent with increased enzyme activity following treatment with IL-27. Inclusion of a soluble receptor to neutralize endogenous IL-27, decreased IDO expression and activity compared with untreated macrophages. In response to IL-27, neonatal macrophages phosphorylate signal transdcuer and activator of transcription 1 (STAT-1) and STAT-3. Both transcription factors are recruited to the IDO regulatory region. STAT-3 dominates during steady-state regulation by lower levels of endogenous IL-27 production. A shift to enhanced STAT-1 recruitment occurs during increased levels of exogenously supplied IL-27. These data suggest an interesting interplay of STAT-1 and STAT-3 to regulate IDO activity and immunosuppression in response to different levels of IL-27 in the microenvironment of the immune response that may further our understanding of this interesting cytokine.

  7. An ancient relative of cyclooxygenase in cyanobacteria is a linoleate 10S-dioxygenase that works in tandem with a catalase-related protein with specific 10S-hydroperoxide lyase activity.

    PubMed

    Brash, Alan R; Niraula, Narayan P; Boeglin, William E; Mashhadi, Zahra

    2014-05-09

    In the course of exploring the scope of catalase-related hemoprotein reactivity toward fatty acid hydroperoxides, we detected a novel candidate in the cyanobacterium Nostoc punctiforme PCC 73102. The immediate neighboring upstream gene, annotated as "cyclooxygenase-2," appeared to be a potential fatty acid heme dioxygenase. We cloned both genes and expressed the cDNAs in Escherichia coli, confirming their hemoprotein character. Oxygen electrode recordings demonstrated a rapid (>100 turnovers/s) reaction of the heme dioxygenase with oleic and linoleic acids. HPLC, including chiral column analysis, UV, and GC-MS of the oxygenated products, identified a novel 10S-dioxygenase activity. The catalase-related hemoprotein reacted rapidly and specifically with linoleate 10S-hydroperoxide (>2,500 turnovers/s) with a hydroperoxide lyase activity specific for the 10S-hydroperoxy enantiomer. The products were identified by NMR as (8E)10-oxo-decenoic acid and the C8 fragments, 1-octen-3-ol and 2Z-octen-1-ol, in ∼3:1 ratio. Chiral HPLC analysis established strict enzymatic control in formation of the 3R alcohol configuration (99% enantiomeric excess) and contrasted with racemic 1-octen-3-ol formed in reaction of linoleate 10S-hydroperoxide with hematin or ferrous ions. The Nostoc linoleate 10S-dioxygenase, the sequence of which contains the signature catalytic sequence of cyclooxygenases and fungal linoleate dioxygenases (YRWH), appears to be a heme dioxygenase ancestor. The novel activity of the lyase expands the known reactions of catalase-related proteins and functions in Nostoc in specific transformation of the 10S-hydroperoxylinoleate.

  8. Preparation of Non-Heme {FeNO}7 Models of Cysteine Dioxygenase: Sulfur Versus Nitrogen Ligation and Photorelease of Nitric Oxide

    PubMed Central

    McQuilken, Alison C.; Ha, Yang; Sutherlin, Kyle D.; Siegler, Maxime A.; Hodgson, Keith O.; Hedman, Britt; Solomon, Edward I.; Jameson, Guy N. L.; Goldberg, David P.

    2013-01-01

    The synthesis and spectroscopic characterization of [Fe(NO)(N3PyS)]BF4 (3) is presented, the first structural and electronic model of NO-bound cysteine dioxygenase (CDO). The nearly isostructural all-N-donor analog [Fe(NO)(N4Py)](BF4)2 (4) was also prepared, and comparisons of 3 and 4 provide insight regarding the influence of S versus N ligation in {FeNO}7 species. One key difference occurs upon photoirradiation, which causes the fully reversible release of NO from 3, but not from 4. PMID:24040838

  9. Replacement of tyrosine 181 by phenylalanine in gentisate 1,2-dioxygenase I from Pseudomonas alcaligenes NCIMB 9867 enhances catalytic activities.

    PubMed

    Tan, Chew Ling; Yeo, Chew Chieng; Khoo, Hoon Eng; Poh, Chit Laa

    2005-11-01

    xlnE, encoding gentisate 1,2-dioxygenase (EC 1.13.11.4), from Pseudomonas alcaligenes (P25X) was mutagenized by site-directed mutagenesis. The mutant enzyme, Y181F, demonstrated 4-, 3-, 6-, and 16-fold increases in relative activity towards gentisate and 3-fluoro-, 4-methyl-, and 3-methylgentisate, respectively. The specific mutation conferred a 13-fold higher catalytic efficiency (k(cat)/Km) on Y181F towards 3-methylgentisate than that of the wild-type enzyme.

  10. Structural Investigations of the Ferredoxin and Terminal Oxygenase Components of the biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae B1

    SciTech Connect

    Ferraro,D.; Brown, E.; Yu, C.; Parales, R.; Gibson, D.; Ramaswamy, S.

    2007-01-01

    The initial step involved in oxidative hydroxylation of monoaromatic and polyaromatic compounds by the microorganism Sphingobium yanoikuyae strain B1 (B1), previously known as Sphingomonas yanoikuyae strain B1 and Beijerinckia sp. strain B1, is performed by a set of multiple terminal Rieske non-heme iron oxygenases. These enzymes share a single electron donor system consisting of a reductase and a ferredoxin (BPDO-F{sub B1}). One of the terminal Rieske oxygenases, biphenyl 2,3-dioxygenase (BPDO-O{sub B1}), is responsible for B1's ability to dihydroxylate large aromatic compounds, such as chrysene and benzo(a)pyrene. Results: In this study, crystal structures of BPDO-O{sub B1} in both native and biphenyl bound forms are described. Sequence and structural comparisons to other Rieske oxygenases show this enzyme to be most similar, with 43.5 % sequence identity, to naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4. While structurally similar to naphthalene 1,2-dioxygenase, the active site entrance is significantly larger than the entrance for naphthalene 1,2-dioxygenase. Differences in active site residues also allow the binding of large aromatic substrates. There are no major structural changes observed upon binding of the substrate. BPDO-F{sub B1} has large sequence identity to other bacterial Rieske ferredoxins whose structures are known and demonstrates a high structural homology; however, differences in side chain composition and conformation around the Rieske cluster binding site are noted. Conclusion: This is the first structure of a Rieske oxygenase that oxidizes substrates with five aromatic rings to be reported. This ability to catalyze the oxidation of larger substrates is a result of both a larger entrance to the active site as well as the ability of the active site to accommodate larger substrates. While the biphenyl ferredoxin is structurally similar to other Rieske ferredoxins, there are distinct changes in the amino acids near the iron

  11. Sequence determination and mutational analysis of the lly locus of Legionella pneumophila.

    PubMed Central

    Wintermeyer, E; Flügel, M; Ott, M; Steinert, M; Rdest, U; Mann, K H; Hacker, J

    1994-01-01

    The lly (legiolysin) locus codes for a 39-kDa protein which confers hemolysis, pigment production, and fluorescence on recombinant Escherichia coli K-12 clones carrying the lly gene. The nucleotide sequences of the lly genes from two Legionella pneumophila isolates were determined. The lly loci exhibited identical nucleotide sequences. They contained open reading frames of 348 amino acid residues, encoding proteins with a deduced molecular mass of 38.9 kDa. N-terminal amino acid sequencing further confirmed that the Lly protein corresponds to the open reading frame sequenced. The amino acid sequence of the Lly protein exhibits a high degree of homology with the sequences of the MelA protein responsible for melanin production in the freshwater bacterium Shewanella colwelliana and the 4-hydroxyphenylpyruvate dioxygenase of Pseudomonas spp. 4-Hydroxyphenylpyruvate dioxygenase is involved in the degradation of aromatic amino acids in various organisms. An Lly-negative mutant of L. pneumophila Philadelphia I derivative JR32 and an Lly-positive transcomplementant were constructed. The Lly-negative mutant lost the ability to produce brown pigment and to confer fluorescence but retained hemolysis. Introduction of a plasmid carrying the lly locus restored pigment production and fluorescence. Intracellular survival of L. pneumophila in U937 macrophage-like cells and in Acanthamoeba castellanii was not affected by mutagenization of the lly locus. Images PMID:8112844

  12. Searching iron sensors in plants by exploring the link among 2′-OG-dependent dioxygenases, the iron deficiency response and metabolic adjustments occurring under iron deficiency

    PubMed Central

    Vigani, Gianpiero; Morandini, Piero; Murgia, Irene

    2013-01-01

    Knowledge accumulated on the regulation of iron (Fe) homeostasis, its intracellular trafficking and transport across various cellular compartments and organs in plants; storage proteins, transporters and transcription factors involved in Fe metabolism have been analyzed in detail in recent years. However, the key sensor(s) of cellular plant “Fe status” triggering the long-distance shoot–root signaling and leading to the root Fe deficiency responses is (are) still unknown. Local Fe sensing is also a major task for roots, for adjusting the internal Fe requirements to external Fe availability: how such sensing is achieved and how it leads to metabolic adjustments in case of nutrient shortage, is mostly unknown. Two proteins belonging to the 2′-OG-dependent dioxygenases family accumulate several folds in Fe-deficient Arabidopsis roots. Such proteins require Fe(II) as enzymatic cofactor; one of their subgroups, the HIF-P4H (hypoxia-inducible factor-prolyl 4-hydroxylase), is an effective oxygen sensor in animal cells. We envisage here the possibility that some members of the 2′-OG dioxygenase family may be involved in the Fe deficiency response and in the metabolic adjustments to Fe deficiency or even in sensing Fe, in plant cells. PMID:23755060

  13. Investigation of acid-base catalysis in the extradiol and intradiol catechol dioxygenase reactions using a broad specificity mutant enzyme and model chemistry.

    PubMed

    Brivio, Michela; Schlosrich, Janne; Ahmad, Mark; Tolond, Caroline; Bugg, Timothy D H

    2009-04-07

    The extradiol and intradiol catechol dioxygenase reaction mechanisms proceed via a common proximal hydroperoxide intermediate, which is processed via different Criegee 1,2-rearrangements. An R215W mutant of extradiol dioxygenase MhpB, able to produce a mixture of extradiol and intradiol cleavage products, was analysed at pH 5.2-8.6, and the yield of extradiol product was found to be highly pH-dependent, whereas the yield of intradiol product was pH-independent. The acid-base chemistry of a biomimetic reaction for extradiol oxidative catechol cleavage was also investigated, using 1,4,7-triazacyclononane, FeCl(2), and pyridine in methanol, in which pyridine is proposed to act as both a general base and (in protonated form) a general acid. Kinetic experiments using a range of meta- and para-substituted pyridines gave a Brønsted plot of log(v) vs. pK(a) showing a bell-shaped plot. Oxidative catechol cleavage by a pyridine-monosubstituted beta-cyclodextrin in the presence of TACN and FeCl(2) in methanol yielded only intradiol cleavage products. It is therefore proposed that bifunctional acid-base catalysis is required for iron (ii)-dependent extradiol catechol cleavage, whereas the rate-determining step for intradiol catechol cleavage does not involve acid-base catalysis.

  14. Effects of Temperature and pH on the Activities of Catechol 2,3-dioxygenase Obtained from Crude Oil Contaminated Soil in Ilaje, Ondo State, Nigeria

    PubMed Central

    Olukunle, O.F.; Babajide, O.; Boboye, B.

    2015-01-01

    Enrichment technique was employed for the isolation of the crude oil degrading bacteria. The isolated bacteria were screened for their degradative ability and the best degrading bacteria were selected based on their growth. Specific activities of Catechol-2,3-dioxygenase and effects of temperature and pH and their stabilities on the enzyme relative activities were observed. Bacteria isolated from the soil sample include; Bacillus cereus, B. amyloliquficiens, B. firmus, Acinetobacter calcoaceticus, Pseudomonas sp. P. fluorescens, P.putida, P.aeruginosa, Achromobacter xylosoxidans and Achromobacter sp. Screening of the degradative ability of the bacteria revealed P. aeruginosa, Bacillus cereus, Acinetobacter calcoaceticus and Achromobacter sp. to be the best degraders. The pH and temperature range with time for the enzyme activity were 6.0-8.0 and 30oC-50oC respectively. The enzyme exhibited activity that was slightly more tolerant to alkaline pH. Therefore, engineering of Catechol 2,3-dioxygenase may be employed for application on bioremediation of polluted sites. PMID:26464607

  15. Non-chemical proton-dependent steps prior to O2-activation limit Azotobacter vinelandii 3-mercaptopropionic acid dioxygenase (MDO) catalysis

    PubMed Central

    Crowell, Joshua K.; Sardar, Sinjinee; Hossain, Mohammad S.; Foss, Frank W.; Pierce, Brad S.

    2016-01-01

    3-mercaptopropionate dioxygenase from Azotobacter vinelandii (Av MDO) is a non-heme mononuclear iron enzyme that catalyzes the O2-dependent oxidation of 3-mercaptopropionate (3mpa) to produce 3-sulfinopropionic acid (3spa). With one exception, the active site residues of MDO are identical to bacterial cysteine dioxygenase (CDO). Specifically, the CDO Arg-residue (R50) is replaced by Gln (Q67) in MDO. Despite this minor active site perturbation, substrate-specificity of Av MDO is more relaxed as compared to CDO. In order to investigate the relative timing of chemical and non-chemical events in Av MDO catalysis, the pH/D-dependence of steady-state kinetic parameters (kcat and kcat/KM) and viscosity effects are measured using two different substrates [3mpa and L-cysteine (cys)]. The pL-dependent activity of Av MDO in these reactions can be rationalized assuming a diprotic enzyme model in which three ionic forms of the enzyme are present [cationic, E(z+1); neutral, Ez; and anionic, E(z−1)]. The activities observed for each substrate appear to be dominated by electrostatic interactions within the enzymatic active site. Given the similarity between MDO and the more extensively characterized mammalian CDO, a tentative model for the role of the conserved ‘catalytic triad’ is proposed. PMID:27311613

  16. Crystallization and preliminary crystallographic analysis of manganese(II)-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase from Bacillus sp. JF8.

    PubMed

    Senda, Miki; Hatta, Takashi; Kimbara, Kazuhide; Senda, Toshiya

    2010-03-01

    A thermostable manganese(II)-dependent 2,3-dihydroxybiphenyl-1,2-dioxygenase derived from Bacillus sp. JF8 was crystallized. The initial screening for crystallization was performed by the sitting-drop vapour-diffusion method using a crystallization robot, resulting in the growth of two crystal forms. The first crystal belonged to space group P1, with unit-cell parameters a = 62.7, b = 71.4, c = 93.6 A, alpha = 71.2, beta = 81.0, gamma = 64.0 degrees, and diffracted to 1.3 A resolution. The second crystal belonged to space group I222, with unit-cell parameters a = 74.2, b = 90.8, c = 104.3 A, and diffracted to 1.3 A resolution. Molecular-replacement trials using homoprotocatechuate 2,3-dioxygenase from Arthrobacter globiformis (28% amino-acid sequence identity) as a search model provided a satisfactory solution for both crystal forms.

  17. Pyruvic oxime dioxygenase from heterotrophic nitrifier Alcaligenes faecalis is a nonheme Fe((II))-dependent enzyme homologous to class II aldolase.

    PubMed

    Tsujino, Shuhei; Uematsu, Chisato; Dohra, Hideo; Fujiwara, Taketomo

    2017-01-06

    Pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification, was purified from Alcaligenes faecalis, and the molecular and catalytic characteristics were reexamined. POD was purified as the homotetramer of the subunit whose molecular weight was 30,000. The deduced amino acid sequence of POD was homologous with a class II aldolase that has been regarded as the Zn((II))-dependent enzyme catalyzing aldol reactions. The recombinant protein showed weak POD activity, and was activated by reconstitution with Fe((II)). Affinity and catalytic constants were estimated at 470 μM and 4.69 sec(-1), respectively. The POD was inactivated by EDTA to remove bound divalent metal cations. A reconstitution experiment demonstrated that Fe((II)), not Zn((II)), is essential for POD activity and that Mn((II)) could partially fulfill the function of Fe((II)). A mutant POD with replacement of His(183), corresponding to one of three Zn((II))-binding ligands in the class II aldolase, by Asn was purified as a homotetrameric protein but showed no catalytic activities. Those results suggest that the POD is homologous to class II aldolase having non-heme Fe((II)) as a catalytic center instead of Zn((II)). A possible mechanism of the POD reaction is discussed on the basis of that of a known Fe((II))-dependent dioxygenase.

  18. Crystal structures of human 3-hydroxyanthranilate 3,4-dioxygenase with native and non-native metals bound in the active site.

    PubMed

    Pidugu, Lakshmi Swarna Mukhi; Neu, Heather; Wong, Tin Lok; Pozharski, Edwin; Molloy, John L; Michel, Sarah L J; Toth, Eric A

    2017-04-01

    3-Hydroxyanthranilate 3,4-dioxygenase (3HAO) is an enzyme in the microglial branch of the kynurenine pathway of tryptophan degradation. 3HAO is a non-heme iron-containing, ring-cleaving extradiol dioxygenase that catalyzes the addition of both atoms of O2 to the kynurenine pathway metabolite 3-hydroxyanthranilic acid (3-HANA) to form quinolinic acid (QUIN). QUIN is a highly potent excitotoxin that has been implicated in a number of neurodegenerative conditions, making 3HAO a target for pharmacological downregulation. Here, the first crystal structure of human 3HAO with the native iron bound in its active site is presented, together with an additional structure with zinc (a known inhibitor of human 3HAO) bound in the active site. The metal-binding environment is examined both structurally and via inductively coupled plasma mass spectrometry (ICP-MS), X-ray fluorescence spectroscopy (XRF) and electron paramagnetic resonance spectroscopy (EPR). The studies identified Met35 as the source of potential new interactions with substrates and inhibitors, which may prove useful in future therapeutic efforts.

  19. Identification and characterization of a novel water-deficit-suppressed gene OsARD encoding an aci-reductone-dioxygenase-like protein in rice.

    PubMed

    Lin, Tao; He, Xiaowei; Yang, Ling; Shou, Huixia; Wu, Ping

    2005-10-24

    The aci-reductone dioxygenase (ARD) family common to bacteria, plants and animals is involved in the methionine salvage pathway. A water-deficit-suppressed gene, OsARD encoding an aci-reductone-dioxygenase-like protein, was identified from rice (Oryza sativa L.). Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the OsARD expression is regulated by abiotic stresses and phytohormones. OsARD was mainly expressed in roots under flood conditions. It was suppressed by abiotic stresses including water deficit, high salinity and low temperature, and induced by ethylene and gibberellin acid (GA). Our results showed that the genes for S-adenosylmethionine (SAM) synthase and 1-aminocyclopropane-1-carboxylic acid (ACC) synthase were upregulated in RNA-interference (RNAi) transgenic rice plants with a significant reduction of OsARD expression. Furthermore, the expression of two genes for ethylene signal transduction, ETR2 and EIN3, increased in these RNAi transgenic plants, whereas the expression of ERF3 was suppressed. These results suggest that OsARD may play a role in the metabolism of methionine and ethylene in response to abiotic stresses.

  20. Characterization of the expression profile of a wheat aci-reductone-dioxygenase-like gene in response to stripe rust pathogen infection and abiotic stresses.

    PubMed

    Xu, Liangsheng; Jia, Jianguang; Lv, Jie; Liang, Xiaofei; Han, Dejun; Huang, Lili; Kang, Zhensheng

    2010-06-01

    The methionine salvage pathway is conserved from prokaryotes to high eukaryotes. The reaction catalyzed by aci-reductone-dioxygenase (ARD) represents a branch point in the methionine salvage pathway. A novel aci-reductone-dioxygenase gene, designed as TaARD, was identified in a subtraction library constructed with RNA isolated from wheat leaves infected with the stripe rust pathogen. TaARD was predicted to encode a 197 amino acid protein that belongs to the cupin superfamily. In transient expression assays with onion epidermal cells, the TaARD-GFP fusion protein localized to the nucleus and cytoplasm. Southern blot analysis showed that the wheat genome had multiple copies of TaARD. Quantitative real-time RT-PCR (qRT-PCR) analyses revealed that the TaARD transcript was induced in wheat leaves infected with a compatible stripe rust strain. However, its expression was reduced or suppressed in incompatible interactions and by ABA, ethephon (ET), or salicylic acid (SA) treatments. With methyl jasmonate (MeJA) treatment, TaARD transcript level was suppressed in the first 6h but increased afterwards. The expression of TaARD also was inhibited by wounding and environmental stimuli, including high salinity and low temperature. Because of the role of ARD in the methionine salvage pathway, these results suggest that TaARD may be involved in ethylene synthesis and ethylene signaling in response to biotic and abiotic stresses.

  1. Searching iron sensors in plants by exploring the link among 2'-OG-dependent dioxygenases, the iron deficiency response and metabolic adjustments occurring under iron deficiency.

    PubMed

    Vigani, Gianpiero; Morandini, Piero; Murgia, Irene

    2013-01-01

    Knowledge accumulated on the regulation of iron (Fe) homeostasis, its intracellular trafficking and transport across various cellular compartments and organs in plants; storage proteins, transporters and transcription factors involved in Fe metabolism have been analyzed in detail in recent years. However, the key sensor(s) of cellular plant "Fe status" triggering the long-distance shoot-root signaling and leading to the root Fe deficiency responses is (are) still unknown. Local Fe sensing is also a major task for roots, for adjusting the internal Fe requirements to external Fe availability: how such sensing is achieved and how it leads to metabolic adjustments in case of nutrient shortage, is mostly unknown. Two proteins belonging to the 2'-OG-dependent dioxygenases family accumulate several folds in Fe-deficient Arabidopsis roots. Such proteins require Fe(II) as enzymatic cofactor; one of their subgroups, the HIF-P4H (hypoxia-inducible factor-prolyl 4-hydroxylase), is an effective oxygen sensor in animal cells. We envisage here the possibility that some members of the 2'-OG dioxygenase family may be involved in the Fe deficiency response and in the metabolic adjustments to Fe deficiency or even in sensing Fe, in plant cells.

  2. Pyruvic oxime dioxygenase from heterotrophic nitrifier Alcaligenes faecalis is a nonheme Fe(II)-dependent enzyme homologous to class II aldolase

    PubMed Central

    Tsujino, Shuhei; Uematsu, Chisato; Dohra, Hideo; Fujiwara, Taketomo

    2017-01-01

    Pyruvic oxime dioxygenase (POD), a key enzyme in heterotrophic nitrification, was purified from Alcaligenes faecalis, and the molecular and catalytic characteristics were reexamined. POD was purified as the homotetramer of the subunit whose molecular weight was 30,000. The deduced amino acid sequence of POD was homologous with a class II aldolase that has been regarded as the Zn(II)-dependent enzyme catalyzing aldol reactions. The recombinant protein showed weak POD activity, and was activated by reconstitution with Fe(II). Affinity and catalytic constants were estimated at 470 μM and 4.69 sec−1, respectively. The POD was inactivated by EDTA to remove bound divalent metal cations. A reconstitution experiment demonstrated that Fe(II), not Zn(II), is essential for POD activity and that Mn(II) could partially fulfill the function of Fe(II). A mutant POD with replacement of His183, corresponding to one of three Zn(II)-binding ligands in the class II aldolase, by Asn was purified as a homotetrameric protein but showed no catalytic activities. Those results suggest that the POD is homologous to class II aldolase having non-heme Fe(II) as a catalytic center instead of Zn(II). A possible mechanism of the POD reaction is discussed on the basis of that of a known Fe(II)-dependent dioxygenase. PMID:28059164

  3. Hydroxylation of aspartic acid in domains homologous to the epidermal growth factor precursor is catalyzed by a 2-oxoglutarate-dependent dioxygenase.

    PubMed Central

    Stenflo, J; Holme, E; Lindstedt, S; Chandramouli, N; Huang, L H; Tam, J P; Merrifield, R B

    1989-01-01

    3-Hydroxyaspartic acid and 3-hydroxyasparagine are two rare amino acids that are present in domains homologous to the epidermal growth factor precursor in vitamin K-dependent plasma proteins as well as in proteins that do not require vitamin K for normal biosynthesis. They are formed by posttranslational hydroxylation of aspartic acid and asparagine, respectively. The first epidermal growth factor-like domain in factor IX (residues 45-87) was synthesized with aspartic acid in position 64, replacing 3-hydroxyaspartic acid. It was used as substrate in a hydroxylase assay with rat liver microsomes as the source of enzyme and reaction conditions that satisfy the requirements of 2-oxoglutarate-dependent dioxygenases. The synthetic peptide stimulated the 2-oxoglutarate decarboxylation in contrast to synthetic, modified epidermal growth factor (Met-21 and His-22 deleted and Glu-24 replaced by Asp) and synthetic peptides corresponding to residues 60-71 in human factor IX. This indicates that the hydroxylase is a 2-oxoglutarate-dependent dioxygenase with a selective substrate requirement. Images PMID:2492106

  4. 3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Pseudomonas aeruginosa: An Fe(II)-containing enzyme with fast turnover.

    PubMed

    Pornsuwan, Soraya; Maenpuen, Somchart; Kamutira, Philaiwarong; Watthaisong, Pratchaya; Thotsaporn, Kittisak; Tongsook, Chanakan; Juttulapa, Maneerat; Nijvipakul, Sarayut; Chaiyen, Pimchai

    2017-01-01

    3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from Pseudomonas aeruginosa (PaDHPAO) was overexpressed in Escherichia coli and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM-1cm-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a Km for DHPA of 58 ± 8 μM and a kcat of 64 s-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(k'cat) versus the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (Ea) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology.

  5. 3,4-Dihydroxyphenylacetate 2,3-dioxygenase from Pseudomonas aeruginosa: An Fe(II)-containing enzyme with fast turnover

    PubMed Central

    Kamutira, Philaiwarong; Watthaisong, Pratchaya; Thotsaporn, Kittisak; Tongsook, Chanakan; Juttulapa, Maneerat; Nijvipakul, Sarayut; Chaiyen, Pimchai

    2017-01-01

    3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from Pseudomonas aeruginosa (PaDHPAO) was overexpressed in Escherichia coli and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM-1cm-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a Km for DHPA of 58 ± 8 μM and a kcat of 64 s-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two pKa values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(k’cat) versus the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (Ea) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology

  6. Protein engineering on biphenyl dioxygenase for conferring activity to convert 7-hydroxyflavone and 5,7-dihydroxyflavone (chrysin).

    PubMed

    Kagami, Osamu; Shindo, Kazutoshi; Kyojima, Akiko; Takeda, Kazuyo; Ikenaga, Hiroshi; Furukawa, Kensuke; Misawa, Norihiko

    2008-08-01

    A central part (amino-acid position 268-397 of 458 amino-acid residues) of the biphenyl dioxygenase large (alpha) subunit, BphA1, from Pseudomonas pseudoalcaligenes strain KF707 was exchanged with the corresponding part of BphA1 from another biphenyl-degrading bacterium, Pseudomonas putida strain KF715, to construct hybrid BphA1, BphA1 (715-707). When expressed in Escherichia coli together with the bphA2A3A4BC genes from strain KF707, this enzyme was shown to possess activity for degrading both 1-phenylnaphthalene and 2-phenylnaphthalene. Between central parts of BphA1 from strains KF707 and KF715, the difference of amino-acid residues resided only in position 324-325. An attempt was made to improve the substrate preference of BphA1 by applying random amino-acid substitutions at these positions to BphA1 (715-707). After screening the mutant library to bioconvert several flavonoids, BphA1 (1-22; T324A and I325L) and BphA1 (2-2; T324L and I325I) were selected. When expressed in E. coli together with bphA2A3A4B from strain KF707, both BphA1 (1-22) and BphA1 (2-2) bioconverted the refractory flavonoids, 7-hydroxyflavone and 5,7-dihydroxyflavone (chrysin), which were hardly converted by any unmodified and artificially-modified shuffled biphenyl dioxygeneses, into their vicinal diol forms, i.e., 2-(2,3-dihydroxyphenyl)-7-hydroxy-chromen-4-one and 2-(2,3-dihydroxyphenyl)-5,7-dihydroxy-chromen-4-one, respectively. In addition, trans-chalcone was converted into 3-(2,3-dihydroxyphenyl)-1-phenylpropan-1-one and further into 1,3-bis-(2,3-dihydroxyphenyl)-propan-1-one. The antioxidative activity of these generated compounds was markedly higher than that of the original substrates used.

  7. Structural, Spectroscopic, and Electrochemical Properties of Nonheme Fe(II)-Hydroquinonate Complexes: Synthetic Models of Hydroquinone Dioxygenases

    PubMed Central

    Baum, Amanda E.; Park, Heaweon; Wang, Denan; Lindeman, Sergey V.; Fiedler, Adam T.

    2012-01-01

    Using the tris(3,5-diphenylpyrazol-1-yl)borate (Ph2Tp) supporting ligand, a series of mono- and dinuclear ferrous complexes containing hydroquinonate (HQate) ligands have been prepared and structurally characterized with X-ray crystallography. The monoiron(II) complexes serve as faithful mimics of the substrate-bound form of hydroquinone dioxygenases (HQDOs) – a family of nonheme Fe enzymes that catalyze the oxidative cleavage of 1,4-dihydroxybenzene units. Reflecting the variety of HQDO substrates, the synthetic complexes feature both mono- and bidentate HQate ligands. The bidentate HQates cleanly provide five-coordinate, high-spin Fe(II) complexes with the general formula [Fe(Ph2Tp)(HLX)] (1X), where HLX is a HQate(1-) ligand substituted at the 2-position with a benzimidazolyl (1A), acetyl (1B and 1C), or methoxy (1D) group. In contrast, the monodentate ligand 2,6-dimethylhydroquinone (H2LF) exhibited a greater tendency to bridge between two Fe(II) centers, resulting in formation of [Fe2(Ph2Tp)2(μ-LF)(MeCN)] [2F(MeCN)]. However, addition of one equivalent of “free” pyrazole (Ph2pz) ligand provided the mononuclear complex, [Fe(Ph2Tp)(HLF)(Ph2pz)] [1F(Ph2pz)], which is stabilized by an intramolecular hydrogen bond between the HLF and Ph2pz donors. Complex 1F(Ph2pz) represents the first crystallographically-characterized example of a monoiron complex bound to an untethered HQate ligand. The geometric and electronic structures of the Fe/HQate complexes were further probed with spectroscopic (UV-vis absorption, 1H NMR) and electrochemical methods. Cyclic voltammograms of complexes in the 1X series revealed an Fe-based oxidation between 0 and −300 mV (vs. Fc+/0), in addition to irreversible oxidation(s) of the HQate ligand at higher potentials. The one-electron oxidized species (1Xox) were examined with UV-vis absorption and electron paramagnetic resonance (EPR) spectroscopies. PMID:22930005

  8. Substrate-Mediated Oxygen Activation by Homoprotocatechuate 2,3-Dioxygenase: Intermediates Formed by a Tyrosine 257 Variant

    PubMed Central

    Mbughuni, Michael M.; Meier, Katlyn K.; Münck, Eckard; Lipscomb, John D.

    2012-01-01

    Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O2 bind in adjacent ligand sites of the active site FeII of Homoprotocatechuate 2,3-Dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the FeII to O2 gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases kcat by about 75%. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed Y257FInt1HPCA and Y257FInt2HPCA, accumulate during the Y257F-HPCA + O2 reaction prior to formation of the ring-cleaved product. Y257FInt1HPCA is colorless and is formed as O2 binds reversibly to the HPCA-enzyme complex. Y257FInt2HPCA forms spontaneously from Y257FInt1HPCA and displays a chromophore at 425 nm (ε425 = 10,500 M-1 cm−1). Mössbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain FeII. The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of FeII, and the low O2 affinity suggests that Y257FInt1HPCA is an HPCA-FeII-O2 complex with little electron delocalization onto the O2. In contrast, the intense spectrum of Y257FInt2HPCA suggests the intermediate is most likely an HPCA quinone-FeII-(hydro)peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA

  9. n-Butilydenephthalide exhibits protection against neurotoxicity through regulation of tryptophan 2, 3 dioxygenase in spinocerebellar ataxia type 3.

    PubMed

    Rajamani, Karthyayani; Liu, Jen-Wei; Wu, Cheng-Han; Chiang, I-Tsang; You, Deng-Huwei; Lin, Si-Yin; Hsieh, Dean-Kuo; Lin, Shinn-Zong; Harn, Horng-Jyh; Chiou, Tzyy-Wen

    2017-02-18

    Spinocerebellar ataxia type 3 or Machado-Joseph disease (SCA3/MJD) is characterized by the repetition of a CAG codon in the ataxin-3 gene (ATXN3), which leads to the formation of an elongated mutant ATXN3 protein that can neither be denatured nor undergo proteolysis in the normal manner. This abnormal proteolysis leads to the accumulation of cleaved fragments, which have been identified as toxic and further they act as a seed for more aggregate formation, thereby increasing toxicity in neuronal cells. To date, there have been few studies or treatment strategies that have focused on controlling toxic fragment formation. The aim of this study is to develop a potential treatment strategy for addressing the complications of toxic fragment formation and to provide an alternative treatment strategy for SCA3. Our preliminary data on anti-aggregation and toxic fragment formation using an HEK (human embryonic kidney cells) 293T-84Q-eGFP (green fluorescent protein) cell model identified n-butilydenephthalide (n-BP) as a potential drug treatment for SCA3. n-BP decreased toxic fragment formation in both SCA3 cell and animal models. Moreover, results showed that n-BP can improve gait, motor coordination, and activity in SCA3 mice. To comprehend the molecular basis behind the control of toxic fragment formation, we used microarray analysis to identify tryptophan metabolism as a major player in controlling the fate of mutant ATXN3 aggregates. We also demonstrated that n-BP functions by regulating the early part of the kynurenine pathway through the downregulation of tryptophan 2, 3-dioxygenase (TDO2), which decreases the downstream neurotoxic product, quinolinic acid (QA). In addition, through the control of TDO2, n-BP also decreases active calpain levels, an important enzyme involved in the proteolysis of mutant ATXN3, thereby decreasing toxic fragment formation and associated neurotoxicity. Collectively, these findings indicate a correlation between n-BP, TDO2, QA, calpain, and

  10. Effects of polycyclic aromatic hydrocarbons on microbial community structure and PAH ring hydroxylating dioxygenase gene abundance in soil.

    PubMed

    Sawulski, Przemyslaw; Clipson, Nicholas; Doyle, Evelyn

    2014-11-01

    Development of successful bioremediation strategies for environments contaminated with recalcitrant pollutants requires in-depth knowledge of the microorganisms and microbial processes involved in degradation. The response of soil microbial communities to three polycyclic aromatic hydrocarbons, phenanthrene (3-ring), fluoranthene (4-ring) and benzo(a)pyrene (5-ring), was examined. Profiles of bacterial, archaeal and fungal communities were generated using molecular fingerprinting techniques (TRFLP, ARISA) and multivariate statistical tools were employed to interpret the effect of PAHs on community dynamics and composition. The extent and rate of PAH removal was directly related to the chemical structure, with the 5-ring PAH benzo(a)pyrene degraded more slowly than phenathrene or fluoranthene. Bacterial, archaeal and fungal communities were all significantly affected by PAH amendment, time and their interaction. Based on analysis of clone libraries, Actinobacteria appeared to dominate in fluoranthene amended soil, although they also represented a significant portion of the diversity in phenanthrene amended and unamended soils. In addition there appeared to be more γ-Proteobacteria and less Bacteroidetes in soil amended with either PAH compared to the control. The soil bacterial community clearly possessed the potential to degrade PAHs as evidenced by the abundance of PAH ring hydroxylating (PAH-RHDα) genes from both gram negative (GN) and gram positive (GP) bacteria in PAH-amended and control soils. Although the dioxygenase gene from GP bacteria was less abundant in soil than the gene associated with GN bacteria, significant (p < 0.001) increases in the abundance of the GP PAH-RHDα gene were observed during phenanthrene and fluoranthene degradation, whereas there was no significant difference in the abundance of the GN PAH-RHDα gene during the course of the experiment. Few studies to-date have examined the effect of pollutants on more than one microbial

  11. Manganese(II) semiquinonato and manganese(III) catecholato complexes with tridentate ligand: modeling the substrate-binding state of manganese-dependent catechol dioxygenase and reactivity with molecular oxygen.

    PubMed

    Komatsuzaki, Hidehito; Shiota, Akihiko; Hazawa, Shogo; Itoh, Muneaki; Miyamura, Noriko; Miki, Nahomi; Takano, Yoichi; Nakazawa, Jun; Inagaki, Akiko; Akita, Munetaka; Hikichi, Shiro

    2013-06-01

    Catecholate catwalk: Monomeric manganese(III) catecholato and manganese(II) semiquinonato complexes as the substrate-binding model of catechol dioxygenase have been synthesized and structurally characterized. The semiquinonato complex reacted with molecular oxygen to give ring-cleaved products and benzoquinone in the catalytic condition.

  12. Structural characterization of 2,6-dichloro-p-hydroquinone 1,2-dioxygenase (PcpA) from Sphingobium chlorophenolicum, a new type of aromatic ring-cleavage enzyme

    PubMed Central

    Hayes, Robert P.; Green, Abigail R.; Nissen, Mark S.; Lewis, Kevin M.; Xun, Luying; Kang, ChulHee

    2014-01-01

    Summary PcpA (2,6-dichloro-p-hydroquinone 1,2-dioxygenase) from Sphingobium chlorophenolicum, a non-haem Fe(II) dioxygenase capable of cleaving the aromatic ring of p-hydroquinone and its substituted variants, is a member of the recently discovered p-hydroquinone 1,2-dioxygenases. Here we report the 2.6 Å structure of PcpA, which consists of four βαβββ motifs, a hallmark of the vicinal oxygen chelate superfamily. The secondary co-ordination sphere of the Fe(II) centre forms an extensive hydrogen-bonding network with three solvent exposed residues, linking the catalytic Fe(II) to solvent. A tight hydrophobic pocket provides p-hydroquinones access to the Fe(II) centre. The p-hydroxyl group is essential for the substrate-binding, thus phenols and catechols, lacking a p-hydroxyl group, do not bind to PcpA. Site-directed mutagenesis and kinetic analysis confirm the critical catalytic role played by the highly conserved His10, Thr13, His226 and Arg259. Based on these results, we propose a general reaction mechanism for p-hydroquinone 1,2-dioxygenases. PMID:23489289

  13. The last step of kanamycin biosynthesis: unique deamination reaction catalyzed by the α-ketoglutarate-dependent nonheme iron dioxygenase KanJ and the NADPH-dependent reductase KanK.

    PubMed

    Sucipto, Hilda; Kudo, Fumitaka; Eguchi, Tadashi

    2012-04-02

    Mystery solved: using heterologous expression, the activities of two enzymes exclusively belonging to the kanamycin biosynthetic pathway have been identified in vitro. A distinctive reaction mechanism to produce kanamycin is proposed and the previously unknown catalytic deamination activity of KanJ dioxygenase is uncovered.

  14. High aromatic ring-cleavage diversity in birch rhizosphere: PAH treatment-specific changes of I.E.3 group extradiol dioxygenases and 16S rRNA bacterial communities in soil.

    PubMed

    Sipilä, Timo P; Keskinen, Anna-Kaisa; Akerman, Marja-Leena; Fortelius, Carola; Haahtela, Kielo; Yrjälä, Kim

    2008-09-01

    Genes encoding key enzymes of catabolic pathways can be targeted by DNA fingerprinting to explore genetic degradation potential in pristine and polluted soils. We performed a greenhouse microcosm experiment to elucidate structural and functional bacterial diversity in polyaromatic hydrocarbon (PAH)-polluted soil and to test the suitability of birch (Betula pendula) for remediation. Degradation of PAHs was analysed by high-performance liquid chromatography, DNA isolated from soil amplified and fingerprinted by restriction fragment length polymorphism (RFLP) and terminal restriction fragment length polymorphism (T-RFLP). Bacterial 16S rRNA T-RFLP fingerprinting revealed a high structural bacterial diversity in soil where PAH amendment altered the general community structure as well as the rhizosphere community. Birch augmented extradiol dioxygenase diversity in rhizosphere showing a rhizosphere effect, and further pyrene was more efficiently degraded in planted pots. Degraders of aromatic compounds upon PAH amendment were shown by the changed extradiol ring-cleavage community structure in soil. The RFLP analysis grouped extradiol dioxygenase marker genes into 17 distinct operational taxonomic units displaying novel phylogenetic clusters of ring-cleavage dioxygenases representing putative catabolic pathways, and the peptide sequences contained conserved amino-acid signatures of extradiol dioxygenases. A branch of major environmental TS cluster was identified as being related to Parvibaculum lavantivorans ring-cleavage dioxygenase. The described structural and functional diversity demonstrated a complex interplay of bacteria in PAH pollution. The findings improve our understanding of rhizoremediation and unveil the extent of uncharacterized enzymes and may benefit bioremediation research by facilitating the development of molecular tools to detect and monitor populations involved in degradative processes.

  15. Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.

    PubMed Central

    Jiang, H; Parales, R E; Lynch, N A; Gibson, D T

    1996-01-01

    The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation. PMID:8655491

  16. Molecular cloning and characterization of desacetoxyvindoline-4-hydroxylase, a 2-oxoglutarate dependent-dioxygenase involved in the biosynthesis of vindoline in Catharanthus roseus (L.) G. Don.

    PubMed

    Vazquez-Flota, F; De Carolis, E; Alarco, A M; De Luca, V

    1997-08-01

    A 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.11) which catalyzes the 4-hydroxylation of desacetoxyvindoline was purified to homogeneity. Three oligopeptides isolated from a tryptic digest of the purified protein were microsequenced and one oligopeptide showed significant homology to hyoscyamine 6 beta-hydroxylase from Hyoscyamus niger. A 36-mer degenerate oligonucleotide based on this peptide sequence was used to screen a Catharanthus roseus cDNA library and three clones, cD4H-1 to -3, were isolated. Although none of the three clones were full-length, the open reading frame on each clone encoded a putative protein containing the sequence of all three peptides. Primer extension analysis suggested that cD4H-3, the longest cDNA clone, was missing 156 bp at the 5' end of the clone and sequencing of the genomic clone, gD4H-8, confirmed these results. Southern blot analysis suggested that d4h is present as a single-copy gene in C. roseus which is a diploid plant, and the significant differences in the sequence of the 3'-UTR between cD4H-1 and -3 suggest that they represent dimorphic alleles of the same hydroxylase. The identity of the clone was further confirmed when extracts of transformed Escherichia coli expressed D4H enzyme activity. The D4H clone encoded a putative protein of 401 amino acids with a calculated molecular mass of 45.5 kDa and the amino acid sequence showed a high degree of similarity with those of a growing family of 2-oxoglutarate-dependent dioxygenases of plant and fungal origin. The similarity was not restricted to the dioxygenase protein sequences but was also extended to the gene structure and organization since the 205 and 1720 bp introns of d4h were inserted around the same highly conserved amino acid consensus sequences as those for e8 protein, hyoscyamine-6 beta-hydroxylase and ethylene-forming enzyme. These results provide further support that a common ancestral gene is responsible for the appearance of this family of dioxygenases

  17. Inhibition of indoleamine 2,3-dioxygenase 1/2 prevented cognitive impairment and energetic metabolism changes in the hippocampus of adult rats subjected to polymicrobial sepsis.

    PubMed

    Comim, Clarissa M; Freiberger, Viviane; Ventura, Letícia; Mina, Francielle; Ferreira, Gabriela K; Michels, Monique; Generoso, Jaqueline S; Streck, Emílio L; Quevedo, João; Barichello, Tatiana; Dal-Pizzol, Felipe

    2017-04-15

    Sepsis is a life-threatening organ dysfunction caused by a dysregulated host response to infection that may affect the brain. We investigated the role of indoleamine 2,3-dioxygenase (IDO-1/2) inhibition on long-term memory and energetic metabolism after experimental sepsis by caecal ligation and perforation (CLP). Experimental sepsis increased the activity of complexes I, II-III and IV at 24h after CLP, and IDO-1/2 inhibition normalized the activity of these complexes in the hippocampus. Wistar rats presented impairment of habituation and aversive memories 10days after CLP. Adjuvant treatment with the IDO inhibitor prevented long-term cognitive impairment triggered by sepsis.

  18. Interferon-γ regulates the proliferation and differentiation of mesenchymal stem cells via activation of indoleamine 2,3 dioxygenase (IDO).

    PubMed

    Croitoru-Lamoury, Juliana; Lamoury, Francois M J; Caristo, Michael; Suzuki, Kazuo; Walker, David; Takikawa, Osamu; Taylor, Rosanne; Brew, Bruce J

    2011-02-16

    The kynurenine pathway (KP) of tryptophan metabolism is linked to antimicrobial activity and modulation of immune responses but its role in stem cell biology is unknown. We show that human and mouse mesenchymal and neural stem cells (MSCs and NSCs) express the complete KP, including indoleamine 2,3 dioxygenase 1 (IDO) and IDO2, that it is highly regulated by type I (IFN-β) and II interferons (IFN-γ), and that its transcriptional modulation depends on the type of interferon, cell type and species. IFN-γ inhibited proliferation and altered human and mouse MSC neural, adipocytic and osteocytic differentiation via the activation of IDO. A functional KP present in MSCs, NSCs and perhaps other stem cell types offers novel therapeutic opportunities for optimisation of stem cell proliferation and differentiation.

  19. Memo is homologous to nonheme iron dioxygenases and binds an ErbB2-derived phosphopeptide in its vestigial active site.

    PubMed

    Qiu, Chen; Lienhard, Susanne; Hynes, Nancy E; Badache, Ali; Leahy, Daniel J

    2008-02-01

    Memo (mediator of ErbB2-driven cell motility) is a 297-amino-acid protein recently shown to co-precipitate with the C terminus of ErbB2 and be required for ErbB2-driven cell motility. Memo is not homologous to any known signaling proteins, and how it mediates ErbB2 signals is not known. To provide a molecular basis for understanding Memo function, we have determined and report here the 2.1A crystal structure of human Memo and show it be homologous to class III nonheme iron-dependent dioxygenases, a structural class that now includes a zinc-binding protein of unknown function. No metal binding or enzymatic activity can be detected for Memo, but Memo does bind directly to a specific ErbB2-derived phosphopeptide encompassing Tyr-1227 using its vestigial enzymatic active site. Memo thus represents a new class of phosphotyrosine-binding protein.

  20. Memo is Homologous to Nonheme Iron Dioxygenases and Binds an ErbB2-Derived Phosphopeptide in its Vestigial Active Site

    SciTech Connect

    Qiu,C.; Lienhard, S.; Hynes, N.; Badache, A.; Leahy, D.

    2008-01-01

    Memo (mediator of ErbB2-driven cell motility) is a 297-amino-acid protein recently shown to co-precipitate with the C terminus of ErbB2 and be required for ErbB2-driven cell motility. Memo is not homologous to any known signaling proteins, and how it mediates ErbB2 signals is not known. To provide a molecular basis for understanding Memo function, we have determined and report here the 2.1A crystal structure of human Memo and show it be homologous to class III nonheme iron-dependent dioxygenases, a structural class that now includes a zinc-binding protein of unknown function. No metal binding or enzymatic activity can be detected for Memo, but Memo does bind directly to a specific ErbB2-derived phosphopeptide encompassing Tyr-1227 using its vestigial enzymatic active site. Memo thus represents a new class of phosphotyrosine-binding protein.

  1. Comparative quantitative prevalence of Mycobacteria and functionally abundant nidA, nahAc, and nagAc dioxygenase genes in coal tar contaminated sediments.

    PubMed

    Debruyn, Jennifer M; Chewning, Christopher S; Sayler, Gary S

    2007-08-01

    The Chattanooga Creek Superfund site is heavily contaminated with metals, pesticides, and coal tar with sediments exhibiting high concentrations of polycyclic aromatic hydrocarbons (PAHs). High molecular weight PAHs are of concern because of their toxicity and recalcitrance in the environment; as such, there is great interest in microbes, such as fast-growing Mycobacterium spp., capable of degradation of these compounds. Real-time quantitative PCR assays were developed targeting multiple dioxygenase genes to assess the ecology and functional diversity of PAH-degrading communities. These assays target the Mycobacterium nidA, beta-proteobacteria nagAc, and gamma-proteobacteria nahAc with the specific goal of testing the hypothesis that Mycobacteria catabolic genes are enriched and may be functionally associated with high molecular weight PAH biodegradation in Chattanooga Creek. Dioxygenase gene abundances were quantitatively compared to naphthalene and pyrene mineralization, and temporal and spatial PAH concentrations. nidA abundances ranged from 5.69 x 10(4) to 4.92 x 10(6) copies per gram sediment; nagAc from 2.42 x 10(3) to 1.21 x 10(7), and nahAc from below detection to 4.01 x 10(6) copies per gram sediment. There was a significantly greater abundance of nidA and nagAc at sites with the greatest concentrations of PAHs. In addition, nidA and nagAc were significantly positively correlated (r = 0.76), indicating a coexistence of organisms carrying these genes. A positive relationship was also observed between nidA and nagAc and pyrene mineralization indicating that these genes serve as biomarkers for pyrene degradation. A 16S rDNA clone library of fast-growing Mycobacteria indicated that the population is very diverse and likely plays an important role in attenuation of high molecular weight PAHs from Chattanooga Creek.

  2. Acireductone dioxygenase- (ARD-) type reactivity of a nickel(II) complex having monoanionic coordination of a model substrate: product identification and comparisons to unreactive analogues.

    PubMed

    Szajna-Fuller, Ewa; Rudzka, Katarzyna; Arif, Atta M; Berreau, Lisa M

    2007-07-09

    A mononuclear Ni(II) complex ([(6-Ph2TPA)Ni(PhC(O)C(OH)C(O)Ph)]ClO4 (1)), supported by the 6-Ph2TPA chelate ligand (6-Ph2TPA = N,N-bis((6-phenyl-2-pyridyl)methyl)-N-((2-pyridyl)methyl)amine) and containing a cis-beta-keto-enolate ligand having a C2 hydroxyl substituent, undergoes reaction with O2 to produce a Ni(II) monobenzoate complex ([(6-Ph2TPA)Ni(O2CPh)]ClO4 (3)), CO, benzil (PhC(O)C(O)Ph), benzoic acid, and other minor unidentified phenyl-containing products. Complex 3 has been identified through independent synthesis and was characterized by X-ray crystallography, 1H NMR, FAB-MS, FTIR, and elemental analysis. A series of cis-beta-keto-enolate Ni(II) complexes supported by the 6-Ph2TPA ligand ([(6-Ph2TPA)Ni(PhC(O)CHC(O)Ph)]ClO4 (4), [(6-Ph2TPA)Ni(CH3C(O)CHC(O)CH3)]ClO4 (5), and [(6-Ph2TPA)Ni(PhC(O)CHC(O)C(O)Ph) (6)) have been prepared and characterized. While these complexes exhibit structural and/or spectroscopic similarity to 1, all are unreactive with O2. The results of this study are discussed in terms of relevance to Ni(II)-containing acireductone dioxygenase enzymes, as well as in the context of recently reported cofactor-free, quercetin, and beta-diketone dioxygenases.

  3. Characterization of the Fungal Gibberellin Desaturase as a 2-Oxoglutarate-Dependent Dioxygenase and Its Utilization for Enhancing Plant Growth1[W][OA

    PubMed Central

    Bhattacharya, Anjanabha; Kourmpetli, Sofia; Ward, Dennis A.; Thomas, Stephen G.; Gong, Fan; Powers, Stephen J.; Carrera, Esther; Taylor, Benjamin; de Caceres Gonzalez, Francisco Nuñez; Tudzynski, Bettina; Phillips, Andrew L.; Davey, Michael R.; Hedden, Peter

    2012-01-01

    The biosynthesis of gibberellic acid (GA3) by the fungus Fusarium fujikuroi is catalyzed by seven enzymes encoded in a gene cluster. While four of these enzymes are characterized as cytochrome P450 monooxygenases, the nature of a fifth oxidase, GA4 desaturase (DES), is unknown. DES converts GA4 to GA7 by the formation of a carbon-1,2 double bond in the penultimate step of the pathway. Here, we show by expression of the des complementary DNA in Escherichia coli that DES has the characteristics of a 2-oxoglutarate-dependent dioxygenase. Although it has low amino acid sequence homology with known 2-oxoglutarate-dependent dioxygenases, putative iron- and 2-oxoglutarate-binding residues, typical of such enzymes, are apparent in its primary sequence. A survey of sequence databases revealed that homologs of DES are widespread in the ascomycetes, although in most cases the homologs must participate in non-gibberellin (GA) pathways. Expression of des from the cauliflower mosaic virus 35S promoter in the plant species Solanum nigrum, Solanum dulcamara, and Nicotiana sylvestris resulted in substantial growth stimulation, with a 3-fold increase in height in S. dulcamara compared with controls. In S. nigrum, the height increase was accompanied by a 20-fold higher concentration of GA3 in the growing shoots than in controls, although GA1 content was reduced. Expression of des was also shown to partially restore growth in plants dwarfed by ectopic expression of a GA 2-oxidase (GA-deactivating) gene, consistent with GA3 being protected from 2-oxidation. Thus, des has the potential to enable substantial growth increases, with practical implications, for example, in biomass production. PMID:22911627

  4. Salicylate 5-Hydroxylase from Ralstonia sp. Strain U2: a Monooxygenase with Close Relationships to and Shared Electron Transport Proteins with Naphthalene Dioxygenase

    PubMed Central

    Zhou, Ning-Yi; Al-Dulayymi, Jumáa; Baird, Mark S.; Williams, Peter A.

    2002-01-01

    The genes from the oxygenase cluster nagAaGHAbAcAd of naphthalene-degrading Ralstonia sp. strain U2 were cloned and overexpressed. Salicylate 5-hydroxylase (S5H) activity, converting salicylate to gentisate, was present in vitro only in the single extract of cells with overexpressed nagAaGHAb or in a mixture of three cell extracts containing, respectively, NagGH (the oxygenase components), NagAa (ferredoxin reductase), and NagAb (ferredoxin). Each of the three extracts required for S5H activity was rate limiting in the presence of excess of the others but, when in excess, did not affect the rate of catalysis. S5H catalyzed the 5-hydroxylation of the aromatic rings of 3- and 4-substituted salicylates. However, the methyl group of 5-methylsalicylate was hydroxylated to produce the 5-hydroxymethyl derivative and the 6-position on the ring of 5-chlorosalicylate was hydroxylated, producing 5-chloro-2,6-dihydroxybenzoate. In an assay for the nag naphthalene dioxygenase (NDO) based on the indole-linked oxidation of NADH, three extracts were essential for activity (NagAcAd, NagAa, and NagAb). NDO and S5H were assayed in the presence of all possible combinations of the nag proteins and the corresponding nah NDO proteins from the “classical” naphthalene degrader P. putida NCIMB9816. All three oxygenase components functioned with mixed combinations of the electron transport proteins from either strain. The S5H from strain U2 is a unique monooxygenase which shares sequence similarity with dioxygenases such as NDO but is also sufficiently similar in structure to interact with the same electron transport chain and probably does so in vivo during naphthalene catabolism in strain U2. PMID:11872705

  5. Mechanism of Repair of Acrolein- and Malondialdehyde-Derived Exocyclic Guanine Adducts by the α-Ketoglutarate/Fe(II) Dioxygenase AlkB

    PubMed Central

    2015-01-01

    The structurally related exocyclic guanine adducts α-hydroxypropano-dG (α-OH-PdG), γ-hydroxypropano-dG (γ-OH-PdG), and M1dG are formed when DNA is exposed to the reactive aldehydes acrolein and malondialdehyde (MDA). These lesions are believed to form the basis for the observed cytotoxicity and mutagenicity of acrolein and MDA. In an effort to understand the enzymatic pathways and chemical mechanisms that are involved in the repair of acrolein- and MDA-induced DNA damage, we investigated the ability of the DNA repair enzyme AlkB, an α-ketoglutarate/Fe(II) dependent dioxygenase, to process α-OH-PdG, γ-OH-PdG, and M1dG in both single- and double-stranded DNA contexts. By monitoring the repair reactions using quadrupole time-of-flight (Q-TOF) mass spectrometry, it was established that AlkB can oxidatively dealkylate γ-OH-PdG most efficiently, followed by M1dG and α-OH-PdG. The AlkB repair mechanism involved multiple intermediates and complex, overlapping repair pathways. For example, the three exocyclic guanine adducts were shown to be in equilibrium with open-ring aldehydic forms, which were trapped using (pentafluorobenzyl)hydroxylamine (PFBHA) or NaBH4. AlkB repaired the trapped open-ring form of γ-OH-PdG but not the trapped open-ring of α-OH-PdG. Taken together, this study provides a detailed mechanism by which three-carbon bridge exocyclic guanine adducts can be processed by AlkB and suggests an important role for the AlkB family of dioxygenases in protecting against the deleterious biological consequences of acrolein and MDA. PMID:25157679

  6. Origin of the Proton-transfer Step in the Cofactor-free (1H)-3-Hydroxy-4-oxoquinaldine 2,4-Dioxygenase

    PubMed Central

    Hernandez-Ortega, Aitor; Quesne, Matthew G.; Bui, Soi; Heuts, Dominic P. H. M.; Steiner, Roberto A.; Heyes, Derren J.; de Visser, Sam P.; Scrutton, Nigel S.

    2014-01-01

    Dioxygenases catalyze a diverse range of chemical reactions that involve the incorporation of oxygen into a substrate and typically use a transition metal or organic cofactor for reaction. Bacterial (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) belongs to a class of oxygenases able to catalyze this energetically unfavorable reaction without any cofactor. In the quinaldine metabolic pathway, HOD breaks down its natural N-heteroaromatic substrate using a mechanism that is still incompletely understood. Experimental and computational approaches were combined to study the initial step of the catalytic cycle. We have investigated the role of the active site His-251/Asp-126 dyad, proposed to be involved in substrate hydroxyl group deprotonation, a critical requirement for subsequent oxygen reaction. The pH profiles obtained under steady-state conditions for the H251A and D126A variants show a strong pH effect on their kcat and kcat/Km constants, with a decrease in kcat/Km of 5500- and 9-fold at pH 10.5, respectively. Substrate deprotonation studies under transient-state conditions show that this step is not rate-limiting and yield a pKa value of ∼7.2 for WT HOD. A large solvent isotope effect was found, and the pKa value was shifted to ∼8.3 in D2O. Crystallographic and computational studies reveal that the mutations have a minor effect on substrate positioning. Computational work shows that both His-251 and Asp-126 are essential for the proton transfer driving force of the initial reaction. This multidisciplinary study offers unambiguous support to the view that substrate deprotonation, driven by the His/Asp dyad, is an essential requirement for its activation. PMID:24482238

  7. Abundance of Dioxygenase Genes Similar to Ralstonia sp. Strain U2 nagAc Is Correlated with Naphthalene Concentrations in Coal Tar-Contaminated Freshwater Sediments

    PubMed Central

    Dionisi, Hebe M.; Chewning, Christopher S.; Morgan, Katherine H.; Menn, Fu-Min; Easter, James P.; Sayler, Gary S.

    2004-01-01

    We designed a real-time PCR assay able to recognize dioxygenase large-subunit gene sequences with more than 90% similarity to the Ralstonia sp. strain U2 nagAc gene (nagAc-like gene sequences) in order to study the importance of organisms carrying these genes in the biodegradation of naphthalene. Sequencing of PCR products indicated that this real-time PCR assay was specific and able to detect a variety of nagAc-like gene sequences. One to 100 ng of contaminated-sediment total DNA in 25-μl reaction mixtures produced an amplification efficiency of 0.97 without evident PCR inhibition. The assay was applied to surficial freshwater sediment samples obtained in or in close proximity to a coal tar-contaminated Superfund site. Naphthalene concentrations in the analyzed samples varied between 0.18 and 106 mg/kg of dry weight sediment. The assay for nagAc-like sequences indicated the presence of (4.1 ± 0.7) × 103 to (2.9 ± 0.3) × 105 copies of nagAc-like dioxygenase genes per μg of DNA extracted from sediment samples. These values corresponded to (1.2 ± 0.6) × 105 to (5.4 ± 0.4) × 107 copies of this target per g of dry weight sediment when losses of DNA during extraction were taken into account. There was a positive correlation between naphthalene concentrations and nagAc-like gene copies per microgram of DNA (r = 0.89) and per gram of dry weight sediment (r = 0.77). These results provide evidence of the ecological significance of organisms carrying nagAc-like genes in the biodegradation of naphthalene. PMID:15240274

  8. Quantitative structure-activity relationship for the cleavage of C3/C4-substituted catechols by a prototypal extradiol catechol dioxygenase with broad substrate specificity.

    PubMed

    Ishida, Tetsuo; Tanaka, Hiroyuki; Horiike, Kihachiro

    2004-06-01

    Catechol 2,3-dioxygenase [EC 1.13.11.2] from Pseudomonas putida mt-2 (Mpc) catalyzes the extradiol cleavage of catechol to produce 2-hydroxymuconate semialdehyde. The K(m) values for the catecholic substrate (K(mA)) and O(2) (K(mO2)), and catalytic constants (k(cat)) were kinetically determined for eight C3/C4-substituted catechols at 25 degrees C and pH 6.5 or 7.5. The first pK(a) values (pK(1)) were determined for eleven catechols (pK(1) = 7.26-9.47), correlated with Hammett substituent constants, and electron-withdrawing substituents significantly stabilized the monoanionic species of free catechols. Mpc preferred catechols with non-ionic substituents at the C3 or C4 position. 3-Phenylcatechol, a biphenyl, was cleaved, while 4-tert-butylcatechol was not. The logarithm of k(cat)/K(mA) (substrate specificity constant) exhibited a good linear correlation with pK(1), with the exception of those for 4-halocatechols. The logarithm of k(cat)/K(mO2) showed a good linear correlation with pK(1), with the exception of that of 3-phenylcatechol. These results demonstrate that catechol binding to the Mpc active site, the following O(2) binding, and the activation of the bound O(2) are all sensitive to electronic effects of the substituents. However, k(cat) did not correlate significantly with pK(1). The present study distinguishes clearly between the electronic and the steric effects of catecholic substrates in the reactivity of Mpc, and provides important insight into the mechanistic basis for a vast range of substrate specificities of extradiol dioxygenases.

  9. Comparative quantitative prevalence of mycobacteria and functionally abundant nidA, nahAc, and nagAc Dioxygenase genes in coal tar contaminated sediments

    SciTech Connect

    Jennifer M. DeBruyn; Christopher S. Chewning; Gary S. Sayler

    2007-08-01

    The Chattanooga Creek Superfund site is heavily contaminated with metals, pesticides, and coal tar with sediments exhibiting high concentrations of polycyclic aromatic hydrocarbons (PAHs). High molecular weight PAHs are of concern because of their toxicity and recalcitrance in the environment; as such, there is great interest in microbes, such as fast-growing Mycobacterium spp., capable of degradation of these compounds. Real-time quantitative PCR assays were developed targeting multiple dioxygenase genes to assess the ecology and functional diversity of PAH-degrading communities. These assays target the Mycobacterium nidA, {beta}-proteobacteria nagAc, and {gamma}-proteobacteria nahAc with the specific goal of testing the hypothesis that Mycobacteria catabolic genes are enriched and may be functionally associated with high molecular weight PAH biodegradation in Chattanooga Creek. Dioxygenase gene abundances were quantitatively compared to naphthalene and pyrene mineralization, and temporal and spatial PAH concentrations. nidA abundances ranged from 5.69 x 10{sup 4} to 4.92 x 10{sup 6} copies per gram sediment; nagAc from 2.42 x 10{sup 3} to 1.21 x 10{sup 7}, and nahAc from below detection to 4.01 x 10{sup 6} copies per gram sediment. There was a significantly greater abundance of nidA and nagAc at sites with the greatest concentrations of PAHs. In addition, nidA and nagAc were significantly positively correlated, indicating a coexistence of organisms carrying these genes. A positive relationship was also observed between nidA and nagAc and pyrene mineralization indicating that these genes serve as biomarkers for pyrene degradation. A 16S rDNA clone library of fast-growing Mycobacteria indicated that the population is very diverse and likely plays an important role in attenuation of high molecular weight PAHs from Chattanooga Creek. 35 refs., 5 figs., 1 tab.

  10. Synthesis of 5-hydroxyectoine from ectoine: crystal structure of the non-heme iron(II) and 2-oxoglutarate-dependent dioxygenase EctD.

    PubMed

    Reuter, Klaus; Pittelkow, Marco; Bursy, Jan; Heine, Andreas; Craan, Tobias; Bremer, Erhard

    2010-05-14

    As a response to high osmolality, many microorganisms synthesize various types of compatible solutes. These organic osmolytes aid in offsetting the detrimental effects of low water activity on cell physiology. One of these compatible solutes is ectoine. A sub-group of the ectoine producer's enzymatically convert this tetrahydropyrimidine into a hydroxylated derivative, 5-hydroxyectoine. This compound also functions as an effective osmostress protectant and compatible solute but it possesses properties that differ in several aspects from those of ectoine. The enzyme responsible for ectoine hydroxylation (EctD) is a member of the non-heme iron(II)-containing and 2-oxoglutarate-dependent dioxygenases (EC 1.14.11). These enzymes couple the decarboxylation of 2-oxoglutarate with the formation of a high-energy ferryl-oxo intermediate to catalyze the oxidation of the bound organic substrate. We report here the crystal structure of the ectoine hydroxylase EctD from the moderate halophile Virgibacillus salexigens in complex with Fe(3+) at a resolution of 1.85 A. Like other non-heme iron(II) and 2-oxoglutarate dependent dioxygenases, the core of the EctD structure consists of a double-stranded beta-helix forming the main portion of the active-site of the enzyme. The positioning of the iron ligand in the active-site of EctD is mediated by an evolutionarily conserved 2-His-1-carboxylate iron-binding motif. The side chains of the three residues forming this iron-binding site protrude into a deep cavity in the EctD structure that also harbours the 2-oxoglutarate co-substrate-binding site. Database searches revealed a widespread occurrence of EctD-type proteins in members of the Bacteria but only in a single representative of the Archaea, the marine crenarchaeon Nitrosopumilus maritimus. The EctD crystal structure reported here can serve as a template to guide further biochemical and structural studies of this biotechnologically interesting enzyme family.

  11. Expression of the Nitroarene Dioxygenase Genes in Comamonas sp. Strain JS765 and Acidovorax sp. Strain JS42 Is Induced by Multiple Aromatic Compounds

    PubMed Central

    Lessner, Daniel J.; Parales, Rebecca E.; Narayan, Shakti; Gibson, David T.

    2003-01-01

    This work reports a genetic analysis of the expression of nitrobenzene dioxygenase (NBDO) in Comamonas sp. strain JS765 and 2-nitrotoluene dioxygenase (2NTDO) in Acidovorax sp. strain JS42. Strains JS765 and JS42 possess identical LysR-type regulatory proteins, NbzR and NtdR, respectively. NbzR/NtdR is homologous to NahR, the positive salicylate-responsive transcriptional activator of the naphthalene degradation genes in Pseudomonas putida G7. The genes encoding NBDO and 2NTDO in each strain are cotranscribed, and transcription starts at the same site within identical promoter regions for each operon. Results from a lacZ reporter gene fusion demonstrated that expression of NBDO and 2NTDO is induced by multiple aromatic compounds, including an array of nitroaromatic compounds (nitrobenzene, 2-, 3-, and 4-nitrotoluene, 2,4- and 2,6-dinitrotoluene, and aminodinitrotoluenes), as well as salicylate and anthranilate. The nitroaromatic compounds appear to be the actual effector molecules. Analysis of β-galactosidase and 2NTDO activities with strain JS42 demonstrated that NtdR was required for induction by all of the inducing compounds, high basal-level expression of 2NTDO, and complementation of a JS42 ntdR null mutant. Complementation with the closely related regulators NagR (from Ralstonia sp. strain U2) and NahR restored only induction by the archetype inducers, salicylate or salicylate and anthranilate, respectively, and did not restore the high basal level of expression of 2NTDO. The mechanism of 2NTDO gene regulation in JS42, and presumably that of NBDO gene regulation in JS765, appear similar to that of NahR-regulated genes in Pseudomonas putida G7. However, NbzR and NtdR appear to have evolved a broader specificity in JS42 and JS765, allowing for recognition of nitroaromatic compounds while retaining the ability to respond to salicylate and anthranilate. NtdR is also the first example of a nitroarene-responsive LysR-type transcriptional activator. PMID:12813084

  12. The c4h, tat, hppr and hppd Genes Prompted Engineering of Rosmarinic Acid Biosynthetic Pathway in Salvia miltiorrhiza Hairy Root Cultures

    PubMed Central

    Gao, Shouhong; Saechao, Saengking; Di, Peng; Chen, Junfeng; Chen, Wansheng

    2011-01-01

    Rational engineering to produce biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Here we capitalized on our previously described gene-to-metabolite network in order to engineer rosmarinic acid (RA) biosynthesis pathway for the production of beneficial RA and lithospermic acid B (LAB) in Salvia miltiorrhiza hairy root cultures. Results showed their production was greatly elevated by (1) overexpression of single gene, including cinnamic acid 4-hydroxylase (c4h), tyrosine aminotransferase (tat), and 4-hydroxyphenylpyruvate reductase (hppr), (2) overexpression of both tat and hppr, and (3) suppression of 4-hydroxyphenylpyruvate dioxygenase (hppd). Co-expression of tat/hppr produced the most abundant RA (906 mg/liter) and LAB (992 mg/liter), which were 4.3 and 3.2-fold more than in their wild-type (wt) counterparts respectively. And the value of RA concentration was also higher than that reported before, that produced by means of nutrient medium optimization or elicitor treatment. It is the first report of boosting RA and LAB biosynthesis through genetic manipulation, providing an effective approach for their large-scale commercial production by using hairy root culture systems as bioreactors. PMID:22242141

  13. Biodegradation of phenanthrene, spatial distribution of bacterial populations and dioxygenase expression in the mycorrhizosphere of Lolium perenne inoculated with Glomus mosseae.

    PubMed

    Corgié, S C; Fons, F; Beguiristain, T; Leyval, C

    2006-05-01

    Interactions between the plant and its microbial communities in the rhizosphere control microbial polycyclic aromatic hydrocarbons (PAH) biodegradation processes. Arbuscular mycorrhizal (AM) fungi can influence plant survival and PAH degradation in polluted soil. This work was aimed at studying the contribution of the mycorrhizosphere to PAH biodegradation in the presence of ryegrass (Lolium perenne L., cv. Barclay) inoculated with Glomus mosseae (BEG 69) by taking into account the structure and activity of bacterial communities, PAH degrading culturable bacteria as a function of the distance from roots. Ryegrass was grown in compartmentalized systems designed to harvest successive sections of rhizosphere in lateral compartments polluted or not with phenanthrene (PHE). Colonization of roots by G. mosseae (BEG 69) modified the structure and density of bacterial populations in the mycorrhizosphere, compared to the rhizosphere of non-mycorrhizal plants. G. mosseae increased the density of culturable heterotrophic and PAH degrading bacteria beyond the immediate rhizosphere in the presence of PHE, and increased the density of PAH degraders in the absence of the pollutant. Biodegradation was not significantly increased in the mycorrhizosphere, compared to control non-mycorrhizal plants, where PHE biodegradation already reached 92% after 6 weeks. However, dioxygenase transcriptional activity was found to be higher in the immediate mycorrhizosphere in the presence of G. mosseae (BEG 69).

  14. An integrated approach of bioassay and molecular docking to study the dihydroxylation mechanism of pyrene by naphthalene dioxygenase in Rhodococcus sp. ustb-1.

    PubMed

    Jin, Jing-Nan; Yao, Jun; Zhang, Qing-Ye; Yu, Chan; Chen, Peng; Liu, Wen-Juan; Peng, Dan-Ning; Choi, Martin M F

    2015-06-01

    Naphthalene dioxygenase (NDO) is the initial enzyme catalyzing the biodegradation of aromatic compounds, and it plays a key role in microbial remediation of polluting sites. In this study, Rhodococcus sp. ustb-1 derived from crude oil was selected to investigate the biodegradation characters and dihydroxylation mechanism of pyrene by an integrated approach of bioassay and molecular docking. The biodegradation experiment proved that the strain ustb-1 shows high effective biodegradability to pyrene with a 70.8% degradation on the 28th day and the metabolite pyrene cis-4,5-dihydrodiol was found. The results of molecular docking indicated that the regions surrounding pyrene are defined by hydrophobic amino acids which are favorable for the binding of dioxygen molecule at C4 and C5 positions of pyrene in a side-on mode. The binding positions of dioxygen are in agreement with the mass spectral analysis of the metabolite pyrene cis-4,5-dihydrodiol. In summary, this study provides a promising explanation for the possible binding behavior between pyrene and active site of NDO.

  15. Umbilical Cord Tissue-Derived Mesenchymal Stem Cells Induce T Lymphocyte Apoptosis and Cell Cycle Arrest by Expression of Indoleamine 2, 3-Dioxygenase

    PubMed Central

    Li, Xiuying; Xu, Zhuo; Bai, Jinping; Yang, Shuyuan; Zhao, Shuli; Zhang, Yingjie; Chen, Xiaodong

    2016-01-01

    It has been reported that human mesenchymal stem cells are able to inhibit T lymphocyte activation; however, the discrepancy among different sources of MSCs is not well documented. In this study, we have compared the MSCs from bone marrow (BM), adipose tissue (AT), placenta (PL), and umbilical cord (UC) to determine which one displayed the most efficient immunosuppressive effects on phytohemagglutinin-induced T cell proliferation. Among them we found that hUC-MSC has the strongest effects on inhibiting T cell proliferation and is chosen to do the further study. We observed that T lymphocyte spontaneously released abundant IFN-γ. And IFN-γ secreted by T lymphocyte could induce the expression of indoleamine 2, 3-dioxygenase (IDO) in hUC-MSCs. IDO was previously reported to induce T lymphocyte apoptosis and cell cycle arrest in S phase. When cocultured with hUC-MSCs, T lymphocyte expression of caspase 3 was significantly increased, while Bcl2 and CDK4 mRNA expression decreased dramatically. Addition of 1-methyl tryptophan (1-MT), an IDO inhibitor, restored T lymphocyte proliferation, reduced apoptosis, and induced resumption of the cell cycle. In addition, the changes in caspase 3, CDK4, and Bcl2 expression were reversed by 1-MT. These findings demonstrate that hUC-MSCs induce T lymphocyte apoptosis and cell cycle arrest by expressing abundant IDO and provide an explanation for some of the immunomodulatory effects of MSCs. PMID:27418932

  16. A preliminary crystallographic study of recombinant NicX, an Fe2+-dependent 2,5-dihydroxypyridine dioxygenase from Pseudomonas putida KT2440

    PubMed Central

    Jiménez, José Ignacio; Acebrón, Iván; García, José Luis; Díaz, Eduardo; Mancheño, José Miguel

    2010-01-01

    NicX from Pseudomonas putida KT2440 is an Fe2+-dependent dioxygenase that is involved in the aerobic degradation of nicotinic acid. The enzyme converts 2,5-­dihydroxypyridine to N-formylmaleamic acid when overexpressed in Escherichia coli. Biophysical characterization of NicX by analytical gel-filtration chromatography revealed that it behaves as an oligomeric assembly in solution, with an apparent molecular weight that is consistent with a hexameric species. NicX was crystallized by the hanging-drop vapour-diffusion method at 291 K. Diffraction data were collected to a resolution of 2.0 Å at the ESRF. The crystals most probably belong to the orthorhombic space group C222 or C2221. The estimated Matthews coefficient was 2.4 Å3 Da−1, corresponding to 50% solvent content, which is consistent with the presence of three protein molecules in the asymmetric unit. Analysis of the crystal data together with chromatographic results supports NicX being a hexameric assembly composed of two cyclic trimers. Currently, crystallization of recombinant selenomethionine-containing NicX is in progress. PMID:20445257

  17. Metabolism and Residues of 2,4-Dichlorophenoxyacetic Acid in DAS-40278-9 Maize (Zea mays) Transformed with Aryloxyalkanoate Dioxygenase-1 Gene.

    PubMed

    Zhou, Xiao; Rotondaro, Sandra L; Ma, Mingming; Rosser, Steve W; Olberding, Ed L; Wendelburg, Brian M; Adelfinskaya, Yelena A; Balcer, Jesse L; Blewett, T Craig; Clements, Bruce

    2016-10-12

    DAS-40278-9 maize, which is developed by Dow AgroSciences, has been genetically modified to express the aryloxyalkanoate dioxygenase-1 (AAD-1) protein and is tolerant to phenoxy auxin herbicides, such as 2,4-dichlorophenoxyacetic acid (2,4-D). To understand the metabolic route and residue distribution of 2,4-D in DAS-40278-9 maize, a metabolism study was conducted with (14)C-radiolabeled 2,4-D applied at the maximum seasonal rate. Plants were grown in boxes outdoors. Forage and mature grain, cobs, and stover were collected for analysis. The metabolism study showed that 2,4-D was metabolized to 2,4-dichlorophenol (2,4-DCP), which was then rapidly conjugated with glucose. Field-scale residue studies with 2,4-D applied at the maximum seasonal rate were conducted at 25 sites in the U.S. and Canada to measure the residues of 2,4-D and free and conjugated 2,4-DCP in mature forage, grain, and stover. Residues of 2,4-D were not detectable in the majority of the grain samples and averaged <1.0 and <1.5 μg/g in forage and stover, respectively. Free plus conjugated 2,4-DCP was not observed in grain and averaged <1.0 μg/g in forage and stover.

  18. Characterization of the promoter region of an Arabidopsis gene for 9-cis-epoxycarotenoid dioxygenase involved in dehydration-inducible transcription.

    PubMed

    Behnam, Babak; Iuchi, Satoshi; Fujita, Miki; Fujita, Yasunari; Takasaki, Hironori; Osakabe, Yuriko; Yamaguchi-Shinozaki, Kazuko; Kobayashi, Masatomo; Shinozaki, Kazuo

    2013-08-01

    Plants respond to dehydration stress and tolerate water-deficit status through complex physiological and cellular processes. Many genes are induced by water deficit. Abscisic acid (ABA) plays important roles in tolerance to dehydration stress by inducing many stress genes. ABA is synthesized de novo in response to dehydration. Most of the genes involved in ABA biosynthesis have been identified, and they are expressed mainly in leaf vascular tissues. Of the products of such genes, 9-cis-epoxycarotenoid dioxygenase (NCED) is a key enzyme in ABA biosynthesis. One of the five NCED genes in Arabidopsis, AtNCED3, is significantly induced by dehydration. To understand the regulatory mechanism of the early stages of the dehydration stress response, it is important to analyse the transcriptional regulatory systems of AtNCED3. In the present study, we found that an overlapping G-box recognition sequence (5'-CACGTG-3') at -2248 bp from the transcriptional start site of AtNCED3 is an important cis-acting element in the induction of the dehydration response. We discuss the possible transcriptional regulatory system of dehydration-responsive AtNCED3 expression, and how this may control the level of ABA under water-deficit conditions.

  19. St. John's Wort increases brain serotonin synthesis by inhibiting hepatic tryptophan 2, 3 dioxygenase activity and its gene expression in stressed rats.

    PubMed

    Bano, Samina; Ara, Iffat; Saboohi, Kausar; Moattar, Tariq; Chaoudhry, Bushra

    2014-09-01

    We aimed to investigate the effects of herbal St. John's Wort (SJW) on transcriptional regulation of hepatic tryptophan 2, 3 - dioxygenase (TDO) enzyme activity and brain regional serotonin (5-HT) levels in rats exposed to forced swim test (FST). TDO mRNA expression was quantified using real-time reverse transcription polymerase chain (RT-PCR) reaction and brain regional indoleamines were determined by high performance liquid chromatography coupled to fluorescence detector. Behavioral analysis shows significant reduction in immobility time in SJW (500mg/kg/ml) administered rats. It was found that pretreatment of SJW to rats did not prevent stress-induced elevation in plasma corticosterone levels however it increases serotonin synthesis by virtue of inhibiting hepatic TDO enzyme activity and its gene expression, ascertaining the notion that there exists an inverse relationship between hepatic TDO enzyme activity and brain 5-HT. The drug also decreases serotonin turnover in all the brain areas (hypothalamus, hippocampus amygdala) in stressed rats endorsing its monoamine oxidase inhibition property. Inhibition of TDO enzyme activity and its gene expression by the drug provides new insights for the development of therapeutic interventions for stress related mental illnesses.

  20. 2-Oxoglutarate-dependent dioxygenases are sensors of energy metabolism, oxygen availability, and iron homeostasis: potential role in the regulation of aging process.

    PubMed

    Salminen, Antero; Kauppinen, Anu; Kaarniranta, Kai

    2015-10-01

    Recent studies have revealed that the members of an ancient family of nonheme Fe(2+)/2-oxoglutarate-dependent dioxygenases (2-OGDO) are involved in the functions associated with the aging process. 2-Oxoglutarate and O2 are the obligatory substrates and Fe(2+) a cofactor in the activation of 2-OGDO enzymes, which can induce the hydroxylation of distinct proteins and the demethylation of DNA and histones. For instance, ten-eleven translocation 1-3 (TET1-3) are the demethylases of DNA, whereas Jumonji C domain-containing histone lysine demethylases (KDM2-7) are the major epigenetic regulators of chromatin landscape, known to be altered with aging. The functions of hypoxia-inducible factor (HIF) prolyl hydroxylases (PHD1-3) as well as those of collagen hydroxylases are associated with age-related degeneration. Moreover, the ribosomal hydroxylase OGFOD1 controls mRNA translation, which is known to decline with aging. 2-OGDO enzymes are the sensors of energy metabolism, since the Krebs cycle intermediate 2-oxoglutarate is an activator whereas succinate and fumarate are the potent inhibitors of 2-OGDO enzymes. In addition, O2 availability and iron redox homeostasis control the activities of 2-OGDO enzymes in tissues. We will briefly elucidate the catalytic mechanisms of 2-OGDO enzymes and then review the potential functions of the above-mentioned 2-OGDO enzymes in the control of the aging process.

  1. Formation of norisoprenoid flavor compounds in carrot (Daucus carota L.) roots: characterization of a cyclic-specific carotenoid cleavage dioxygenase 1 gene.

    PubMed

    Yahyaa, Mosaab; Bar, Einat; Dubey, Neeraj Kumar; Meir, Ayala; Davidovich-Rikanati, Rachel; Hirschberg, Joseph; Aly, Radi; Tholl, Dorothea; Simon, Philipp W; Tadmor, Yaakov; Lewinsohn, Efraim; Ibdah, Mwafaq

    2013-12-18

    Carotenoids are isoprenoid pigments that upon oxidative cleavage lead to the production of norisoprenoids that have profound effect on flavor and aromas of agricultural products. The biosynthetic pathway to norisoprenoids in carrots (Daucus carota L.) is still largely unknown. We found the volatile norisoprenoids farnesylacetone, α-ionone, and β-ionone accumulated in Nairobi, Rothild, and Purple Haze cultivars but not in Yellowstone and Creme de Lite in a pattern reflecting their carotenoid content. A cDNA encoding a protein with carotenoid cleavage dioxygenase activity, DcCCD1, was identified in carrot and was overexpressed in Escherichia coli strains previously engineered to produce different carotenoids. The recombinant DcCCD1 enzyme cleaves cyclic carotenes to generate α- and β-ionone. No cleavage products were found when DcCCD1 was co-expressed in E. coli strains accumulating non-cyclic carotenoids, such as phytoene or lycopene. Our results suggest a role for DcCCD1 in carrot flavor biosynthesis.

  2. Insights from the predicted structural analysis of carborane substituted withaferin A with Indoleamine - 2,3-dioxygenase as a potent inhibitor

    PubMed Central

    Samad, Firoz A

    2016-01-01

    Indoleamine-2,3-dioxygenase (IDO) an immunoregulatory enzyme and emerging as a new therapeutic drug target for the treatment of cancer. Carboranes, an icosahedral arrangement of eleven boron atoms plus one carbon atom with unique pharmacological properties such low toxicity, isosterism with phenyl ring and stability to hydrolysis. On the other hand, carboranes are known to increase the interaction of ligand with non-polar region of the protein provides an excellent platform to explore these carboranes towards designing and development of novel, potent and target specific drug candidates with further enhanced binding affinities. Despite of their many potential applications, molecular modeling studies of carborane-substituted ligands with macromolecules have been rarely reported. Previously, we have demonstrated the promising high binding affinity of Withaferin-A (WA) for IDO. In this present study, we investigated the effect of carborane substitutions on WA compound towards developing novel analogs for target specific IDO inhibition with better potency. Interesting docked poses and molecular interactions for the carborane substituted WA ligands were elucidated. Based on our In-silico studies, carborane substituted at various position of WA has shown enhanced binding affinity towards IDO, worth of considering for further studies. PMID:28250615

  3. Oral Probiotic VSL#3 Prevents Autoimmune Diabetes by Modulating Microbiota and Promoting Indoleamine 2,3-Dioxygenase-Enriched Tolerogenic Intestinal Environment.

    PubMed

    Dolpady, Jayashree; Sorini, Chiara; Di Pietro, Caterina; Cosorich, Ilaria; Ferrarese, Roberto; Saita, Diego; Clementi, Massimo; Canducci, Filippo; Falcone, Marika

    2016-01-01

    The gut microbiota modulates the autoimmune pathogenesis of type 1 diabetes (T1D) via mechanisms that remain largely unknown. The inflammasome components are innate immune sensors that are highly influenced by the gut environment and play pivotal roles in maintaining intestinal immune homeostasis. In this study we show that modifications of the gut microbiota induced by oral treatment with Lactobacillaceae-enriched probiotic VSL#3, alone or in combination with retinoic acid (RA), protect NOD mice from T1D by affecting inflammasome at the intestinal level. In particular, we show that VSL#3 treatment inhibits IL-1β expression while enhancing release of protolerogenic components of the inflammasome, such as indoleamine 2,3-dioxygenase (IDO) and IL-33. Those modifications of the intestinal microenvironment in VSL#3-treated NOD mice modulate gut immunity by promoting differentiation of tolerogenic CD103(+) DCs and reducing differentiation/expansion of Th1 and Th17 cells in the intestinal mucosa and at the sites of autoimmunity, that is, within the pancreatic lymph nodes (PLN) of VSL#3-treated NOD mice. Our data provide a link between dietary factors, microbiota composition, intestinal inflammation, and immune homeostasis in autoimmune diabetes and could pave the way for new therapeutic approaches aimed at changing the intestinal microenvironment with probiotics to counterregulate autoimmunity and prevent T1D.

  4. Identification of an evolutionary conserved structural loop that is required for the enzymatic and biological function of tryptophan 2,3-dioxygenase

    PubMed Central

    Michels, Helen; Seinstra, Renée I.; Uitdehaag, Joost C. M.; Koopman, Mandy; van Faassen, Martijn; Martineau, Céline N.; Kema, Ido P.; Buijsman, Rogier; Nollen, Ellen A. A.

    2016-01-01

    The enzyme TDO (tryptophan 2,3-dioxygenase; TDO-2 in Caenorhabditis elegans) is a potential therapeutic target to cancer but is also thought to regulate proteotoxic events seen in the progression of neurodegenerative diseases. To better understand its function and develop specific compounds that target TDO we need to understand the structure of this molecule. In C. elegans we compared multiple different CRISPR/Cas9-induced tdo-2 deletion mutants and identified a motif of three amino acids (PLD) that is required for the enzymatic conversion of tryptophan to N-formylkynurenine. Loss of TDO-2’s enzymatic activity in PDL deletion mutants was accompanied by an increase in motility during aging and a prolonged lifespan, which is in line with the previously observed phenotypes induced by a knockdown of the full enzyme. Comparison of sequence structures suggests that blocking this motif might interfere with haem binding, which is essential for the enzyme’s activity. The fact that these three residues are situated in an evolutionary conserved structural loop of the enzyme suggests that the findings can be translated to humans. The identification of this specific loop region in TDO-2–essential for its catalytic function–will aid in the design of novel inhibitors to treat diseases in which the TDO enzyme is overexpressed or hyperactive. PMID:27995966

  5. Membrane-type 1 matrix metalloproteinase cytoplasmic tail binding protein-1 (MTCBP-1) acts as an eukaryotic aci-reductone dioxygenase (ARD) in the methionine salvage pathway.

    PubMed

    Hirano, Wakako; Gotoh, Isamu; Uekita, Takamasa; Seiki, Motoharu

    2005-06-01

    MTCBP-1 was identified as a protein that binds the cytoplasmic tail of membrane-type 1 matrix metalloproteinase (MT1-MMP/MMP-14). Since MTCBP-1 has a putative beta-barrel structure, it is presumably a member of the recently proposed cupin superfamily that contains tremendously diverged functions of proteins in spite of their well-conserved beta-barrel structure. MTCBP-1 shows significant homology to the bacterial aci-reductone dioxygenase (ARD) in the cupin family, which is an enzyme in the methionine salvage pathway (MTA cycle). Since it is difficult to speculate the functions of cupin proteins simply based on their sequence homology, we examined whether the eukaryotic ARD homologs surely function in the methionine metabolism. Under sulfur-depleted conditions, yeast could grow when substrate of MTA cycle was provided. Disruption of the yeast ARD homolog, YMR009w gene, abolished ability of the cells to grow in this culture condition. Re-expression of either the YMR009w or MTCBP-1 gene restored the cell growth. Mutation analysis revealed that the glutamic acid residue in the beta-barrel fold and the N-terminal extension from the beta-barrel fold were found to be important for the activity to restore the growth. Thus, MTCBP-1 isolated as a binding protein for MT1-MMP was demonstrated to function as an ARD-like enzyme in the MTA cycle in yeast.

  6. The role of indoleamine 2,3-dioxygenase-aryl hydrocarbon receptor pathway in the TLR4-induced tolerogenic phenotype in human DCs

    PubMed Central

    Salazar, Fabián; Awuah, Dennis; Negm, Ola H.; Shakib, Farouk; Ghaemmaghami, Amir M.

    2017-01-01

    A controlled inflammatory response is required for protection against infection, but persistent inflammation causes tissue damage. Dendritic cells (DCs) have a unique capacity to promote both inflammatory and anti-inflammatory processes. One key mechanism involved in DC-mediated immunosuppression is the expression of tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase (IDO). IDO has been implicated in diverse processes in health and disease but its role in endotoxin tolerance in human DCs is still controversial. Here we investigated the role of IDO in shaping DCs phenotype and function under endotoxin tolerance conditions. Our data show that TLR4 ligation in LPS-primed DCs, induced higher levels of both IDO isoforms together with the transcription factor aryl-hydrocarbon receptor (AhR), compared to unprimed controls. Additionally, LPS conditioning induced an anti-inflammatory phenotype in DCs - with an increase in IL-10 and higher expression of programmed death ligand (PD-L)1 and PD-L2 - which were partially dependent on IDO. Furthermore, we demonstrated that the AhR-IDO pathway was responsible for the preferential activation of non-canonical NF-κB pathway in LPS-conditioned DCs. These data provide new insight into the mechanisms of the TLR4-induced tolerogenic phenotype in human DCs, which can help the better understanding of processes involved in induction and resolution of chronic inflammation and tolerance. PMID:28256612

  7. Co-delivery of indoleamine 2,3-dioxygenase prevents loss of expression of an antigenic transgene in dystrophic mouse muscles.

    PubMed

    Sharma, D; Al-Khalidi, R; Edgar, S; An, Q; Wang, Y; Young, C; Nowis, D; Gorecki, D C

    2017-02-01

    A significant problem affecting gene therapy approaches aiming at achieving long-term transgene expression is the immune response against the protein product of the therapeutic gene, which can reduce or eliminate the therapeutic effect. The problem is further exacerbated when therapy involves targeting an immunogenic tissue and/or one with a pre-existing inflammatory phenotype, such as dystrophic muscles. In this proof-of-principle study, we co-expressed a model antigen, bacterial β-galactosidase, with an immunosuppressive factor, indoleamine 2,3-dioxygenase 1 (IDO1), in muscles of the mdx mouse model of Duchenne muscular dystrophy. This treatment prevented loss of expression of the transgene concomitant with significantly elevated expression of T-regulatory (Treg) markers in the IDO1-expressing muscles. Moreover, co-expression of IDO1 resulted in reduced serum levels of anti-β-gal antibodies. These data indicate that co-expression of genes encoding immunomodulatory enzymes controlling kynurenine pathways provide a viable strategy for preventing loss of transgenes targeted into dystrophic muscles with pre-existing inflammation.

  8. Enhanced tolerance and remediation to mixed contaminates of PCBs and 2,4-DCP by transgenic alfalfa plants expressing the 2,3-dihydroxybiphenyl-1,2-dioxygenase.

    PubMed

    Wang, Yan; Ren, Hejun; Pan, Hongyu; Liu, Jinliang; Zhang, Lanying

    2015-04-09

    Polychlorinated biphenyls (PCBs) and 2,4-dichlorophenol (2,4-DCP) generally led to mixed contamination of soils as a result of commercial and agricultural activities. Their accumulation in the environment poses great risks to human and animal health. Therefore, the effective strategies for disposal of these pollutants are urgently needed. In this study, genetic engineering to enhance PCBs/2,4-DCP phytoremediation is a focus. We cloned the 2,3-dihydroxybiphenyl-1,2-dioxygenase (BphC.B) from a soil metagenomic library, which is the key enzyme of aerobic catabolism of a variety of aromatic compounds, and then it was expressed in alfalfa driven by CaMV 35S promoter using Agrobacterium-mediated transformation. Transgenic line BB11 was selected out through PCR, Western blot analysis and enzyme activity assays. Its disposal and tolerance to both PCBs and 2,4-DCP were examined. The tolerance capability of transgenic line BB11 towards complex contaminants of PCBs/2,4-DCP significantly increased compared with non-transgenic plants. Strong dissipation of PCBs and high removal efficiency of 2,4-DCP were exhibited in a short time. It was confirmed expressing BphC.B would be a feasible strategy to help achieving phytoremediation in mixed contaminated soils with PCBs and 2,4-DCP.

  9. Inhibition of indoleamine 2,3-dioxygenase activity enhances the anti-tumour effects of a Toll-like receptor 7 agonist in an established cancer model.

    PubMed

    Ito, Hiroyasu; Ando, Tatsuya; Arioka, Yuko; Saito, Kuniaki; Seishima, Mitsuru

    2015-04-01

    Toll-like receptor (TLR) agonists have been shown to have anti-tumour activity in basic research and clinical studies. However, TLR agonist monotherapy does not sufficiently eliminate tumours. Activation of the innate immune response by TLR agonists is effective at driving adaptive immunity via interleukin-12 (IL-12) or IL-1, but is counteracted by the simultaneous induction of immunosuppressive cytokines and other molecules, including IL-10, transforming growth factor-β, and indoleamine 2,3-dioxygenase (IDO). In the present study, we evaluated the anti-cancer effect of the TLR7 agonist, imiquimod (IMQ), in the absence of IDO activity. The administration of IMQ in IDO knockout (KO) mice inoculated with tumour cells significantly suppressed tumour progression compared with that in wild-type (WT) mice, and improved the survival rate. Moreover, injection with IMQ enhanced the tumour antigen-specific T helper type 1 response in IDO-KO mice with tumours. Combination therapy with IMQ and an IDO inhibitor also significantly inhibited tumour growth. Our results indicated that the enhancement of IDO expression with TLR agonists in cancer treatment might impair host anti-tumour immunity while the inhibition of IDO could enhance the therapeutic efficacy of TLR agonists via the increase of T helper type 1 immune response.

  10. Early Carcinogenesis Involves the Establishment of Immune Privilege via Intrinsic and Extrinsic Regulation of Indoleamine 2,3-dioxygenase-1: Translational Implications in Cancer Immunotherapy

    PubMed Central

    Holtzhausen, Alisha; Zhao, Fei; Evans, Kathy S.; Hanks, Brent A.

    2014-01-01

    Although prolonged genetic pressure has been conjectured to be necessary for the eventual development of tumor immune evasion mechanisms, recent work is demonstrating that early genetic mutations are capable of moonlighting as both intrinsic and extrinsic modulators of the tumor immune microenvironment. The indoleamine 2,3-dioxygenase-1 (IDO) immunoregulatory enzyme is emerging as a key player in tumor-mediated immune tolerance. While loss of the tumor suppressor, BIN-1, and the over-expression of cyclooxygenase-2 have been implicated in intrinsic regulation of IDO, recent findings have demonstrated the loss of TβRIII and the upregulation of Wnt5a by developing cancers to play a role in the extrinsic control of IDO activity by local dendritic cell populations residing within tumor and tumor-draining lymph node tissues. Together, these genetic changes are capable of modulating paracrine signaling pathways in the early stages of carcinogenesis to establish a site of immune privilege by promoting the differentiation and activation of local regulatory T cells. Additional investigation of these immune evasion pathways promises to provide opportunities for the development of novel strategies to synergistically enhance the efficacy of the evolving class of T cell-targeted “checkpoint” inhibitors. PMID:25339948

  11. Salmonella-Based Therapy Targeting Indoleamine 2,3-Dioxygenase Coupled with Enzymatic Depletion of Tumor Hyaluronan Induces Complete Regression of Aggressive Pancreatic Tumors

    PubMed Central

    Manuel, Edwin R.; Chen, Jeremy; D'Apuzzo, Massimo; Lampa, Melanie G.; Kaltcheva, Teodora I.; Thompson, Curtis B.; Ludwig, Thomas; Chung, Vincent; Diamond, Don J.

    2015-01-01

    Bacterial-based therapies are emerging as effective cancer treatments and hold promise for refractory neoplasms such as pancreatic ductal adenocarcinoma (PDAC), which has not shown significant improvement in therapy for over twenty-five years. Using a novel combination of shIDO-ST, a Salmonella-based therapy targeting the immunosuppressive molecule indoleamine 2,3-dioxygenase (IDO), with an enzyme, PEGPH20, which depletes extracellular matrix hyaluronan, we observed extended survival with frequent total regression of autochthonous and orthotopic PDAC tumors. This was associated with migration and accumulation of activated polymorphonuclear neutrophils (PMN) from spleens into tumors, which was not observed using a scrambled control (shScr-ST). Purified splenic PMNs from PEGPH20/shIDO-ST-treated mice exhibited significant IDO knockdown and were able to kill tumor targets ex-vivo through mechanisms involving FasL and serine proteases. In addition, CD8+ T cells were observed to contribute to late control of pancreatic tumors. Collectively, our data demonstrate that entry of shIDO-ST and PMNs into otherwise impermeable desmoplastic tumors is facilitated by PEGPH20-mediated HA removal, further highlighting an important component of effective treatment for PDAC. PMID:26134178

  12. Insight into the metabolism of 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (DDT) by biphenyl dioxygenases.

    PubMed

    L'Abbée, José-Bruno; Tu, Youbin; Barriault, Diane; Sylvestre, Michel

    2011-12-01

    In this work we have investigated the ability of the biphenyl dioxygenase of Burkholderia xenovorans LB400 (BphAE(LB400)) and of Pandoraea pnomenusa B356 (BphAE(B356)) to metabolize DDT. Data show BphAE(LB400) is unable to metabolize this substrate but BphAE(B356) metabolizes DDT to produce two stereoisomers. Structural analysis of DDT-docked BphAE(LB400) and BphAE(B356) identified residue Phe336 of BphAE(LB400) as critical to prevent productive binding of DDT to BphAE(LB400). Furthermore, the fact that residue Gly319 of BphAE(B356) is less constrained than Gly321 of BphAE(LB400) most likely contributes to the ability of BphAE(B356) to bind DDT productively. This was confirmed by examining the ability of BphAE chimeras obtained by shuffling bphA genes from strain B356 and LB400. Chimeras where residues Thr335 (which modulates the constraints on Gly321) and Phe336 (which contacts the substrate) of BphAE(LB400) were replaced by Gly and Ile respectively were able to metabolize DDT. However their stereospecificities varied depending on the presence of other segments or residues from BphAE(B356). Structural analysis suggests that either one or both of residue 267 and a segments comprised of residue 247-260 are likely involved in stereospecificity.

  13. Oral Probiotic VSL#3 Prevents Autoimmune Diabetes by Modulating Microbiota and Promoting Indoleamine 2,3-Dioxygenase-Enriched Tolerogenic Intestinal Environment

    PubMed Central

    Dolpady, Jayashree; Sorini, Chiara; Di Pietro, Caterina; Cosorich, Ilaria; Ferrarese, Roberto; Saita, Diego; Clementi, Massimo; Falcone, Marika

    2016-01-01

    The gut microbiota modulates the autoimmune pathogenesis of type 1 diabetes (T1D) via mechanisms that remain largely unknown. The inflammasome components are innate immune sensors that are highly influenced by the gut environment and play pivotal roles in maintaining intestinal immune homeostasis. In this study we show that modifications of the gut microbiota induced by oral treatment with Lactobacillaceae-enriched probiotic VSL#3, alone or in combination with retinoic acid (RA), protect NOD mice from T1D by affecting inflammasome at the intestinal level. In particular, we show that VSL#3 treatment inhibits IL-1β expression while enhancing release of protolerogenic components of the inflammasome, such as indoleamine 2,3-dioxygenase (IDO) and IL-33. Those modifications of the intestinal microenvironment in VSL#3-treated NOD mice modulate gut immunity by promoting differentiation of tolerogenic CD103+ DCs and reducing differentiation/expansion of Th1 and Th17 cells in the intestinal mucosa and at the sites of autoimmunity, that is, within the pancreatic lymph nodes (PLN) of VSL#3-treated NOD mice. Our data provide a link between dietary factors, microbiota composition, intestinal inflammation, and immune homeostasis in autoimmune diabetes and could pave the way for new therapeutic approaches aimed at changing the intestinal microenvironment with probiotics to counterregulate autoimmunity and prevent T1D. PMID:26779542

  14. Targeting myeloid-derived suppressor cells with colony stimulating factor-1 receptor blockade can reverse immune resistance to immunotherapy in indoleamine 2,3-dioxygenase-expressing tumors

    PubMed Central

    Holmgaard, Rikke B.; Zamarin, Dmitriy; Lesokhin, Alexander; Merghoub, Taha; Wolchok, Jedd D.

    2016-01-01

    Tumor indoleamine 2,3-dioxygenase (IDO) promotes immunosuppression by direct action on effector T cells and Tregs and through recruitment, expansion and activation of myeloid-derived suppressor cells (MDSCs). Targeting of MDSCs is clinically being explored as a therapeutic strategy, though optimal targeting strategies and biomarkers predictive of response are presently unknown. Maturation and tumor recruitment of MDSCs are dependent on signaling through the receptor tyrosine kinase CSF-1R on myeloid cells. Here, we show that MDSCs are the critical cell population in IDO-expressing B16 tumors in mediating accelerated tumor outgrowth and resistance to immunotherapy. Using a clinically relevant drug, we show that inhibition of CSF-1R signaling can functionally block tumor-infiltrating MDSCs and enhance anti-tumor T cell responses. Furthermore, inhibition of CSF-1R sensitizes IDO-expressing tumors to immunotherapy with T cell checkpoint blockade, and combination of CSF-1R blockade with IDO inhibitors potently elicits tumor regression. These findings provide evidence for a critical and functional role for MDSCs on the in vivo outcome of IDO-expressing tumors. PMID:27211548

  15. Protocatechuate 3,4-dioxygenase: a wide substrate specificity enzyme isolated from Stenotrophomonas maltophilia KB2 as a useful tool in aromatic acid biodegradation.

    PubMed

    Guzik, Urszula; Hupert-Kocurek, Katarzyna; Sitnik, Małgorzata; Wojcieszyńska, Danuta

    2014-01-01

    Protocatechuate 3,4-dioxygenases (P34Os) catalyze the reaction of the ring cleavage of aromatic acid derivatives. It is a key reaction in many xenobiotic metabolic pathways. P34Os characterize narrow substrate specificity. This property is an unfavorable feature in the biodegradation process because one type of pollution is rarely present in the environment. Thus, the following study aimed at the characterization of a P34O from Stenotrophomonas maltophilia KB2, being able to utilize a wide spectrum of aromatic carboxylic acids. A total of 3 mM vanillic acid and 4-hydroxybenzoate were completely degraded during 8 and 4.5 h, respectively. When cells of strain KB2 were grown on 9 mM 4-hydroxybenzoate, P34O was induced. Biochemical analysis revealed that the examined enzyme was similar to other known P34Os, but showed untypical wide substrate specificity. A high activity of P34O against 2,4- and 3,5-dihydroxybenzoate was observed. As these substrates do not possess ortho configuration hydroxyl groups, it is postulated that their cleavage could be connected with their monodentate binding of substrate to the active site. Since this enzyme characterizes untypical wide substrate specificity it makes it a useful tool in applications for environmental clean-up purposes.

  16. Tomato carotenoid cleavage dioxygenases 1A and 1B: Relaxed double bond specificity leads to a plenitude of dialdehydes, mono-apocarotenoids and isoprenoid volatiles

    PubMed Central

    Ilg, Andrea; Bruno, Mark; Beyer, Peter; Al-Babili, Salim

    2014-01-01

    The biosynthetic processes leading to many of the isoprenoid volatiles released by tomato fruits are still unknown, though previous reports suggested a clear correlation with the carotenoids contained within the fruit. In this study, we investigated the activity of the tomato (Solanum lycopersicum) carotenoid cleavage dioxygenase (SlCCD1B), which is highly expressed in fruits, and of its homolog SlCCD1A. Using in vitro assays performed with purified recombinant enzymes and by analyzing products formed by the two enzymes in carotene-accumulating Escherichia coli strains, we demonstrate that SlCCD1A and, to a larger extent, SlCCD1B, have a very relaxed specificity for both substrate and cleavage site, mediating the oxidative cleavage of cis- and all-trans-carotenoids as well as of different apocarotenoids at many more double bonds than previously reported. This activity gives rise to a plenitude of volatiles, mono-apocarotenoids and dialdehyde products, including cis-pseudoionone, neral, geranial, and farnesylacetone. Our results provide a direct evidence for a carotenoid origin of these compounds and point to CCD1s as the enzymes catalyzing the formation of the vast majority of tomato isoprenoid volatiles, many of which are aroma constituents. PMID:25057464

  17. Silencing of an α-dioxygenase gene, Ca-DOX, retards growth and suppresses basal disease resistance responses in Capsicum annum.

    PubMed

    Hong, Chi Eun; Ha, Young-Im; Choi, Hyoju; Moon, Ju Yeon; Lee, Jiyoung; Shin, Ah-Young; Park, Chang Jin; Yoon, Gyeong Mee; Kwon, Suk-Yoon; Jo, Ick-Hyun; Park, Jeong Mee

    2017-03-01

    Alpha-dioxygenases (α-DOX) catalyzing the primary oxygenation of fatty acids to oxylipins were recently found in plants. Here, the biological roles of the pepper α-DOX (Ca-DOX) gene, which is strongly induced during non-host pathogen infection in chili pepper, were examined. Virus-induced gene silencing demonstrated that down-regulation of Ca-DOX enhanced susceptibility to bacterial pathogens and suppressed the hypersensitive response via the suppression of pathogenesis-related genes such as PR4, proteinase inhibitor II and lipid transfer protein (PR14). Ca-DOX-silenced pepper plants also exhibited more retarded growth with lower epidermal cell numbers and reduced cell wall thickness than control plants. To better understand regulation of Ca-DOX, transgenic Arabidopsis plants harboring the β-glucuronidase (GUS) reporter gene driven from a putative Ca-DOX promoter were generated. GUS expression was significantly induced upon avirulent pathogen infection in transgenic Arabidopsis leaves, whereas GUS induction was relatively weak upon virulent pathogen treatment. After treatment with plant hormones, early and strong GUS expression was seen after treatment of salicylic acid, whereas ethylene and methyl jasmonate treatments produced relatively weak and late GUS signals. These results will enable us to further understand the role of α-DOX, which is important in lipid metabolism, defense responses, and growth development in plants.

  18. Single amino acid substitution in homogentisate 1,2-dioxygenase is responsible for pigmentation in a subset of Burkholderia cepacia complex isolates

    PubMed Central

    Gonyar, Laura A.; Fankhauser, Sarah C.; Goldberg, Joanna B.

    2015-01-01

    Summary The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacilli that are ubiquitous in the environment and have emerged over the past 30 years as opportunistic pathogens in immunocompromised populations, specifically individuals with cystic fibrosis (CF) and chronic granulomatous disease. This complex of at least 18 distinct species is phenotypically and genetically diverse. One phenotype observed in a subset of Burkholderia cenocepacia (a prominent Bcc pathogen) isolates is the ability to produce a melanin-like pigment. Melanins have antioxidant properties and have been shown to act as virulence factors allowing pathogens to resist killing by the host immune system. The melanin-like pigment expressed by B. cenocepacia is produced through tyrosine catabolism, specifically through the autoxidation and polymerization of homogentisate. Burkholderia cenocepacia J2315 is a CF clinical isolate that displays a pigmented phenotype when grown under normal laboratory conditions. We examined the amino acid sequences of critical enzymes in the melanin synthesis pathway in pigmented and non-pigmented Bcc isolates, and found that an amino acid substitution of glycine for arginine at amino acid 378 in homogentisate 1,2-dioxygenase correlated with pigment production; we identify this as one mechanism for expression of pigment in Bcc isolates. PMID:25294803

  19. Single amino acid substitution in homogentisate 1,2-dioxygenase is responsible for pigmentation in a subset of Burkholderia cepacia complex isolates.

    PubMed

    Gonyar, Laura A; Fankhauser, Sarah C; Goldberg, Joanna B

    2015-04-01

    The Burkholderia cepacia complex (Bcc) is a group of Gram-negative bacilli that are ubiquitous in the environment and have emerged over the past 30 years as opportunistic pathogens in immunocompromised populations, specifically individuals with cystic fibrosis (CF) and chronic granulomatous disease. This complex of at least 18 distinct species is phenotypically and genetically diverse. One phenotype observed in a subset of Burkholderia cenocepacia (a prominent Bcc pathogen) isolates is the ability to produce a melanin-like pigment. Melanins have antioxidant properties and have been shown to act as virulence factors allowing pathogens to resist killing by the host immune system. The melanin-like pigment expressed by B. cenocepacia is produced through tyrosine catabolism, specifically through the autoxidation and polymerization of homogentisate. Burkholderia cenocepacia J2315 is a CF clinical isolate that displays a pigmented phenotype when grown under normal laboratory conditions. We examined the amino acid sequences of critical enzymes in the melanin synthesis pathway in pigmented and non-pigmented Bcc isolates, and found that an amino acid substitution of glycine for arginine at amino acid 378 in homogentisate 1,2-dioxygenase correlated with pigment production; we identify this as one mechanism for expression of pigment in Bcc isolates.

  20. Chimeric Vaccine Stimulation of Human Dendritic Cell Indoleamine 2, 3-Dioxygenase Occurs via the Non-Canonical NF-κB Pathway.

    PubMed

    Kim, Nan-Sun; Mbongue, Jacques C; Nicholas, Dequina A; Esebanmen, Grace E; Unternaehrer, Juli J; Firek, Anthony F; Langridge, William H R

    2016-01-01

    A chimeric protein vaccine composed of the cholera toxin B subunit fused to proinsulin (CTB-INS) was shown to suppress type 1 diabetes onset in NOD mice and upregulate biosynthesis of the tryptophan catabolic enzyme indoleamine 2, 3-dioxygenase (IDO1) in human dendritic cells (DCs). Here we demonstrate siRNA inhibition of the NF-κB-inducing kinase (NIK) suppresses vaccine-induced IDO1 biosynthesis as well as IKKα phosphorylation. Chromatin immunoprecipitation (ChIP) analysis of CTB-INS inoculated DCs showed that RelB bound to NF-κB consensus sequences in the IDO1 promoter, suggesting vaccine stimulation of the non-canonical NF-κB pathway activates IDO1 expression in vivo. The addition of Tumor Necrosis Factor Associated Factors (TRAF) TRAF 2, 3 and TRAF6 blocking peptides to vaccine inoculated DCs was shown to inhibit IDO1 biosynthesis. This experimental outcome suggests vaccine activation of the TNFR super-family receptor pathway leads to upregulation of IDO1 biosynthesis in CTB-INS inoculated dendritic cells. Together, our experimental data suggest the CTB-INS vaccine uses a TNFR-dependent signaling pathway of the non-canonical NF-κB signaling pathway resulting in suppression of dendritic cell mediated type 1 diabetes autoimmunity.

  1. SAHA down-regulates the expression of indoleamine 2,3-dioxygenase via inhibition of the JAK/STAT1 signaling pathway in gallbladder carcinoma cells.

    PubMed

    Zhang, Peng; Jiang, Guanmin; Gao, Jiao; Li, Lingling; Du, Jun; Jiao, Xingyuan

    2013-01-01

    The aim of the present study was to investigate the role of the JAK/STAT1 signaling pathway in suberoylanilide hydroxamic acid (SAHA)-mediated down-regulation of indoleamine 2,3-dioxygenase (IDO) in gallbladder carcinoma cells. We treated SGC-996 gallbladder carcinoma cells with IFN-γ and SAHA. Western blotting was used to detect the expression of IDO, signal transducer and activator of transcription 1 (STAT1) phosphorylation and interferon regulatory factor genes-1 (IRF-1). Confocal microscopy analysis was used to detect STAT1 translocation. Transient transfection and reporter gene assay was used for detecting the activation of γ-activated sites (GAS) and interferon-stimulated response elements (ISRE). The results revealed that IDO was expressed in SGC-996 cells in a dose- and time-dependent manner when stimulated with IFN-γ and SAHA down-regulated the expression of IDO induced by IFN-γ in a dose-dependent manner. SAHA blocked the expression of IRF-1 induced by IFN-γ and SAHA inhibited IFN-γ-induced STAT1 phosphorylation and nuclear translocation. In addition, SAHA down-regulated IFN-γ-induced activation of GAS and ISRE. In conclusion, SAHA down-regulated IDO expression via inhibition of the activation of members of the JAK/STAT1 signaling pathway. Therefore, regulation of the JAK/STAT1 signaling pathway may provide a new gallbladder carcinoma immunotherapeutic strategy to break tumor immune tolerance.

  2. Isolation and preliminary characterization of the subunits of the terminal component of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4.

    PubMed Central

    Suen, W C; Gibson, D T

    1993-01-01

    The terminal oxygenase component (ISPNAP) of naphthalene dioxygenase from Pseudomonas putida NCIB 9816-4 was purified to homogeneity. The protein contained approximately 4 g-atoms each of iron and acid-labile sulfide per mol of ISPNAP, and enzyme activity was stimulated significantly by addition of exogenous iron. The large (alpha) and small (beta) subunits of ISPNAP were isolated by two different procedures. The NH2-terminal amino acid sequences of the alpha and beta subunits were identical to the deduced amino acid sequences reported for the ndoB and ndoC genes from P. putida NCIB 9816 and almost identical to the NH2-terminal amino acid sequences determined for the large and small subunits of ISPNAP from P. putida G7. Gel filtration in the presence of 6 M urea gave an alpha subunit with an absorption maximum at 325 nm and broad absorption between 420 and 450 nm. The alpha subunit contained approximately 2 g-atoms each of iron and acid-labile sulfide per mol of the subunit. The beta subunit did not contain iron or acid-labile sulfide. These results, taken in conjunction with the deduced amino acid sequences of the large subunits from several iron-sulfur oxygenases, indicate that each alpha subunit of ISPNAP contains a Rieske [2Fe-2S] center. Images PMID:8376335

  3. Engineering catechol 1, 2-dioxygenase by design for improving the performance of the cis, cis-muconic acid synthetic pathway in Escherichia coli

    PubMed Central

    Han, Li; Liu, Pi; Sun, Jixue; Wu, Yuanqing; Zhang, Yuanyuan; Chen, Wujiu; Lin, Jianping; Wang, Qinhong; Ma, Yanhe

    2015-01-01

    Regulating and ameliorating enzyme expression and activity greatly affects the performance of a given synthetic pathway. In this study, a new synthetic pathway for cis, cis-muconic acid (ccMA) production was reconstructed without exogenous induction by regulating the constitutive expression of the important enzyme catechol 1,2-dioxygenase (CatA). Next, new CatAs with significantly improved activities were developed to enhance ccMA production using structure-assisted protein design. Nine mutations were designed, simulated and constructed based on the analysis of the CatA crystal structure. These results showed that mutations at Gly72, Leu73 and/or Pro76 in CatA could improve enzyme activity, and the activity of the most effective mutant was 10-fold greater than that of the wild-type CatA from Acinetobacter sp. ADP1. The most productive synthetic pathway with a mutated CatA increased the titer of ccMA by more than 25%. Molecular dynamic simulation results showed that enlarging the entrance of the substrate-binding pocket in the mutants contributed to their increased enzyme activities and thus improved the performance of the synthetic pathway. PMID:26306712

  4. MhNCED3, a gene encoding 9-cis-epoxycarotenoid dioxygenase in Malus hupehensis Rehd., enhances plant tolerance to Cl- stress by reducing Cl- accumulation.

    PubMed

    Zhang, Wei-wei; Yang, Hong-qiang; You, Shu-zhen; Fan, Shu-lei; Ran, Kun

    2015-04-01

    High Cl(-) concentrations in tissues can be toxic to crop plants and may lead to reduced growth rates and yields. 9-cis-epoxycarotenoid dioxygenase (NCED) is thought to be involved in the biosynthesis of abscisic acid (ABA), which is an important regulator of plant adaptive responses to stress. Here, the expression of MhNCED3 in Malus hupehensis Rehd. and the effects of MhNCED3 on plant tolerance to Cl(-) stress were explored. The results showed that MhNCED3 expression and ABA biosynthesis in M. hupehensis Rehd. were induced by Cl(-) stress. Ectopic expression of MhNCED3 in Arabidopsis complemented the phenotypic defects of the 129B08/nced3 mutant and enhanced WT tolerance to Cl(-) stress. The transgenic Arabidopsis showed improved growth and developmental status, increased ABA contents, and reduced transpiration rates and relative water content. Furthermore, ectopic expression of MhNCED3 decreased Cl(-) accumulation and oxidative damage, and up-regulated the expression levels of AtCLCc (chloride channel protein) and AtSLAH3 (slow anion channel 1 homolog 3) genes in Arabidopsis. These observations suggest that MhNCED3 has critical role in enhancing plant tolerance to Cl(-) stress by reducing Cl(-) accumulation.

  5. Eosinophil Granulocytes Account for Indoleamine 2,3-Dioxygenase-Mediated Immune Escape in Human Non-Small Cell Lung Cancer1

    PubMed Central

    Astigiano, Simonetta; Morandi, Barbara; Costa, Roberta; Mastracci, Luca; D'Agostino, Antonella; Battista Ratto, Giovanni; Melioli, Giovanni; Frumento, Guido

    2005-01-01

    Abstract Indoleamine 2,3-dioxygenase (IDO), a catabolizing enzyme of tryptophan, is supposed to play a role in tumor immune escape. Its expression in solid tumors has not yet been well elucidated: IDO can be expressed by the tumor cells themselves, or by ill-defined infiltrating cells, possibly depending on tumor type. We have investigated IDO expression in 25 cases of non-small cell lung cancer (NSCLC). Using histochemistry and immunohistochemistry, we found that IDO was expressed not by tumor cells, but by normal cells infiltrating the peritumoral stroma. These cells were neither macrophages nor dendritic cells, and were identified as eosinophil granulocytes. The amount of IDO-positive eosinophils varied in different cases, ranging from a few cells to more than 50 per field at x200 magnification. IDO protein in NSCLC was enzymatically active. Therefore, at least in NSCLC cases displaying a large amount of these cells in the inflammatory infiltrate, IDO-positive eosinophils could exert an effective immunosuppressive action. On analyzing the 17 patients with adequate follow-up, a significant relationship was found between the amount of IDO-positive infiltrate and overall survival. This finding suggests that the degree of IDO-positive infiltrate could be a prognostic marker in NSCLC. PMID:15967116

  6. Intracerebroventricular Administration of Streptozotocin as an Experimental Approach to Depression: Evidence for the Involvement of Proinflammatory Cytokines and Indoleamine-2,3-Dioxygenase.

    PubMed

    Souza, Leandro Cattelan; Jesse, Cristiano R; de Gomes, Marcelo Gomes; Viana, Cristini Escobar; Mattos, Etiara; Silva, Neici Cáceres; Boeira, Silvana Peterini

    2017-02-02

    There is a lack of information about the molecular events underlying the depressive-like effect of an intracerebroventricular injection of streptozotocin (ICV-STZ) in mice. Elevated activity of the tryptophan-degrading enzyme indoleamine-2,3-dioxygenase (IDO) has been proposed to mediate depression in inflammatory disorders. In this study, we report that ICV-STZ activates IDO in the hippocampus of mice and culminates in depressive-like behaviors, measured by an increased duration in immobility time in the forced swimming test and decreased total time of grooming in the splash test. Indirect blockade of IDO activation with the cytokine inhibitor minocycline prevents the development of depressive-like behaviors and attenuates STZ-induced upregulation of proinflammatory cytokines in the hippocampus. Minocycline abrogates the increase in tryptophan and kynurenine levels as well as prevents serotonin dysfunction in the hippocampus of STZ-injected mice. These results suggest that hippocampal IDO activation in STZ-associated depressive-like behavior is mediated by proinflammatory cytokine-dependent mechanisms. Our study not only extends the evidence that IDO has a critical role in mediating inflammation-induced depression but also supports the notion that neuroinflammation and the kynurenine pathway are important targets of novel therapeutic drugs for depression. In addition, our study provides new insights into the neurobiological mechanisms underlying ICV-STZ and indicates that this model could be employed in the preclinical research of depression.

  7. Developmental and stress regulation of gene expression for a 9-cis-epoxycarotenoid dioxygenase, CstNCED, isolated from Crocus sativus stigmas.

    PubMed

    Ahrazem, Oussama; Rubio-Moraga, Angela; Trapero, Almudena; Gómez-Gómez, Lourdes

    2012-01-01

    Oxidative cleavage of cis-epoxycarotenoids by 9-cis-epoxycarotenoid dioxygenase (NCED) is the critical step in the regulation of abscisic acid (ABA) synthesis in higher plants. ABA has been associated with dormancy and flower senescence, while also regulating plant adaptive responses to various environmental stresses. An NCED gene, CstNCED, was cloned from Crocus sativus stigmas. The deduced amino acid sequence of the CstNCED protein shared high identity with other monocot NCEDs, and was closely related to the liliopsida enzymes. At the N-terminus of CstNCED a chloroplast transit peptide sequence is located. However, its expression in chloroplast-free tissues suggested localization in other plastid types. The relationship between expression of CstNCED and the endogenous ABA level was investigated in the stigma and corms, where it was developmentally regulated. The senescence of the unpollinated stigma is preceded by an increase in ABA levels and CstNCED expression. In corms, a correlation was observed between CstNCED expression and dormancy. Furthermore, CstNCED expression was correlated with the presence of zeaxanthin in the dormant corms. When detached C. sativus leaves and stigmas were water and salt stressed, increases in CstNCED mRNA were observed. The results provided evidence of the involvement of CstNCED in the regulation of ABA-associated processes such as flower senescence and corm dormancy in monocotyledonous saffron.

  8. Genomic analysis and gene structure of the plant carotenoid dioxygenase 4 family: a deeper study in Crocus sativus and its allies.

    PubMed

    Ahrazem, Oussama; Trapero, Almudena; Gómez, M Dolores; Rubio-Moraga, Angela; Gómez-Gómez, Lourdes

    2010-10-01

    The plastoglobule-targeted enzyme carotenoid cleavage dioxygenase (CCD4) mediates the formation of volatile C13 ketones, such as β-ionone, by cleaving the C9-C10 and C9'-C10' double bonds of cyclic carotenoids. Here, we report the isolation and analysis of CCD4 genomic DNA regions in Crocus sativus. Different CCD4 alleles have been identified: CsCCD4a which is found with and without an intron and CsCCD4b that showed the presence of a unique intron. The presence of different CCD4 alleles was also observed in other Crocus species. Furthermore, comparison of the locations of CCD4 introns within the coding region with CCD4 genes from other plant species suggests that independent gain/losses have occurred. The comparison of the promoter region of CsCCD4a and CsCCD4b with available CCD4 gene promoters from other plant species highlighted the conservation of cis-elements involved in light response, heat stress, as well as the absence and unique presence of cis-elements involved in circadian regulation and low temperature responses, respectively. Functional characterization of the Crocus sativus CCD4a promoter using Arabidopsis plants stably transformed with a DNA fragment of 1400 base pairs (P-CsCCD4a) fused to the β-glucuronidase (GUS) reporter gene showed that this sequence was sufficient to drive GUS expression in the flower, in particular high levels were detected in pollen.

  9. The Fe-heme structure of met-indoleamine 2,3-dioxygenase-2 determined by X-ray absorption fine structure

    SciTech Connect

    Aitken, Jade B.; Austin, Christopher J.D.; Hunt, Nicholas H.; Ball, Helen J.; Lay, Peter A.

    2014-07-18

    Highlights: • IDO2 is a newly discovered tryptophan metabolising enzyme with a role in immunity. • IDO2’s active site contains a heme moiety for tryptophan binding and catabolism. • EXAFS/XANES analysis provides the first data of an IDO2 Fe-heme environment. • IDO2 Fe-heme exists as a low spin bis(His) form at 10 K; mixed spin-state at RT. - Abstract: Multiple-scattering (MS) analysis of EXAFS data on met-indoleamine 2,3-dioxygenase-2 (IDO2) and analysis of XANES have provided the first direct structural information about the axial donor ligands of the iron center for this recently discovered protein. At 10 K, it exists in a low-spin bis(His) form with Fe–N{sub p}(av) = 1.97 Å, the Fe–N{sub Im} bond lengths of 2.11 Å and 2.05 Å, which is in equilibrium with a high-spin form at room temperature. The bond distances in the low-spin form are consistent with other low-spin hemeproteins, as is the XANES spectrum, which is closer to that of the low-spin met-Lb than that of the high-spin met-Mb. The potential physiological role of this spin equilibrium is discussed.

  10. Expression of 9-cis-EPOXYCAROTENOID DIOXYGENASE4 Is Essential for Thermoinhibition of Lettuce Seed Germination but Not for Seed Development or Stress Tolerance[C][W

    PubMed Central

    Huo, Heqiang; Dahal, Peetambar; Kunusoth, Keshavulu; McCallum, Claire M.; Bradford, Kent J.

    2013-01-01

    Thermoinhibition, or failure of seeds to germinate at warm temperatures, is common in lettuce (Lactuca sativa) cultivars. Using a recombinant inbred line population developed from a lettuce cultivar (Salinas) and thermotolerant Lactuca serriola accession UC96US23 (UC), we previously mapped a quantitative trait locus associated with thermoinhibition of germination to a genomic region containing a gene encoding a key regulated enzyme in abscisic acid (ABA) biosynthesis, 9-cis-EPOXYCAROTENOID DIOXYGENASE4 (NCED4). NCED4 from either Salinas or UC complements seeds of the Arabidopsis thaliana nced6-1 nced9-1 double mutant by restoring germination thermosensitivity, indicating that both NCED4 genes encode functional proteins. Transgenic expression of Salinas NCED4 in UC seeds resulted in thermoinhibition, whereas silencing of NCED4 in Salinas seeds led to loss of thermoinhibition. Mutations in NCED4 also alleviated thermoinhibition. NCED4 expression was elevated during late seed development but was not required for seed maturation. Heat but not water stress elevated NCED4 expression in leaves, while NCED2 and NCED3 exhibited the opposite responses. Silencing of NCED4 altered the expression of genes involved in ABA, gibberellin, and ethylene biosynthesis and signaling pathways. Together, these data demonstrate that NCED4 expression is required for thermoinhibition of lettuce seeds and that it may play additional roles in plant responses to elevated temperature. PMID:23503626

  11. Circadian Regulation of the PhCCD1 Carotenoid Cleavage Dioxygenase Controls Emission of β-Ionone, a Fragrance Volatile of Petunia Flowers1

    PubMed Central

    Simkin, Andrew J.; Underwood, Beverly A.; Auldridge, Michele; Loucas, Holly M.; Shibuya, Kenichi; Schmelz, Eric; Clark, David G.; Klee, Harry J.

    2004-01-01

    Carotenoids are thought to be the precursors of terpenoid volatile compounds that contribute to flavor and aroma. One such volatile, β-ionone, is important to fragrance in many flowers, including petunia (Petunia hybrida). However, little is known about the factors regulating its synthesis in vivo. The petunia genome contains a gene encoding a 9,10(9′,10′) carotenoid cleavage dioxygenase, PhCCD1. The PhCCD1 is 94% identical to LeCCD1A, an enzyme responsible for formation of β-ionone in tomato (Lycopersicon esculentum; Simkin AJ, Schwartz SH, Auldridge M, Taylor MG, Klee HJ [2004] Plant J [in press]). Reduction of PhCCD1 transcript levels in transgenic plants led to a 58% to 76% decrease in β-ionone synthesis in the corollas of selected petunia lines, indicating a significant role for this enzyme in volatile synthesis. Quantitative reverse transcription-PCR analysis revealed that PhCCD1 is highly expressed in corollas and leaves, where it constitutes approximately 0.04% and 0.02% of total RNA, respectively. PhCCD1 is light-inducible and exhibits a circadian rhythm in both leaves and flowers. β-Ionone emission by flowers occurred principally during daylight hours, paralleling PhCCD1 expression in corollas. The results indicate that PhCCD1 activity and β-ionone emission are likely regulated at the level of transcript. PMID:15516502

  12. Discovery and evaluation of inhibitors to the immunosuppressive enzyme indoleamine 2,3-dioxygenase 1 (IDO1): Probing the active site-inhibitor interactions.

    PubMed

    Tomek, Petr; Palmer, Brian D; Flanagan, Jack U; Sun, Chuanwen; Raven, Emma L; Ching, Lai-Ming

    2017-01-27

    High expression of the immunosuppressive enzyme, indoleamine 2,3-dioxygenase 1 (IDO1) for a broad range of malignancies is associated with poor patient prognosis, and the enzyme is a validated target for cancer intervention. To identify novel IDO1 inhibitors suitable for drug development, 1597 compounds in the National Cancer Institute Diversity Set III library were tested for inhibitory activity against recombinant human IDO1. We retrieved 35 hits that inhibited IDO1 activity >50% at 20 μM. Five structural filters and the PubChem Bioassay database were used to guide the selection of five inhibitors with IC50 between 3 and 12 μM for subsequent experimental evaluation. A pyrimidinone scaffold emerged as being the most promising. It showed excellent cell penetration, negligible cytotoxicity and passed four out of the five structural filters applied. To evaluate the importance of Ser167 and Cys129 residues in the IDO1 active site for inhibitor binding, the entire NCI library was subsequently screened against alanine-replacement mutant enzymes of these two residues. The results established that Ser167 but not Cys129 is important for inhibitory activity of a broad range of IDO1 inhibitors. Structure-activity-relationship studies proposed substituents interacting with Ser167 on four investigated IDO1 inhibitors. Three of these four Ser167 interactions associated with an increased IDO1 inhibition and were correctly predicted by molecular docking supporting Ser167 as an important mediator of potency for IDO1 inhibitors.

  13. Tryptamine and dimethyltryptamine inhibit indoleamine 2,3 dioxygenase and increase the tumor-reactive effect of peripheral blood mononuclear cells.

    PubMed

    Tourino, Melissa Cavalheiro; de Oliveira, Edson Mendes; Bellé, Luziane Potrich; Knebel, Franciele Hinterholz; Albuquerque, Renata Chaves; Dörr, Felipe Augusto; Okada, Sabrina Sayori; Migliorini, Silene; Soares, Irene Silva; Campa, Ana

    2013-07-01

    Indoleamine 2,3-dioxygenase (IDO) is an interferon-γ (IFN-γ)-induced tryptophan-degrading enzyme, producing kynurenine (KYN) that participates in the mechanism of tumor immune tolerance. Thus, IDO inhibition has been considered a strategy for anticancer therapy. The aim of this study was to identify whether the metabolites originated from the competitive routes of tryptophan metabolism, such as the serotonergic or N, N-dimethyltryptamine (DMT) pathways, have inhibitory effects on recombinant human IDO (rhIDO) activity. Serotonin and melatonin had no effect; on the other hand, tryptamine (TRY) and DMT modulated the activity of rhIDO as classical non-competitive inhibitors, with Ki values of 156 and 506 μM, respectively. This inhibitory effect was also observed on constitutively expressed or IFN-γ-induced IDO in the A172 human glioma cell line. TRY and DMT increased the cytotoxic activity of peripheral blood mononuclear cells (PBMCs) in co-culture assays. We conclude that the IDO inhibition by TRY and DMT contributed to a more effective tumor-reactive response by the PBMCs.

  14. Indoleamine 2,3-dioxygenase regulates anti-tumor immunity in lung cancer by metabolic reprogramming of immune cells in the tumor microenvironment

    PubMed Central

    Schafer, Cara C.; Wang, Yong; Hough, Kenneth P.; Sawant, Anandi; Grant, Stefan C.; Thannickal, Victor J.; Zmijewski, Jaroslaw; Ponnazhagan, Selvarangan; Deshane, Jessy S.

    2016-01-01

    Indoleamine 2,3-dioxygenase (IDO) has been implicated in immune evasion by tumors. Upregulation of this tryptophan (Trp)-catabolizing enzyme, in tumor cells and myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment (TME), leads to Trp depletion that impairs cytotoxic T cell responses and survival; however, exact mechanisms remain incompletely understood. We previously reported that a combination therapy of gemcitabine and a superoxide dismutase mimetic promotes anti-tumor immunity in a mouse model of lung cancer by inhibiting MDSCs, enhancing polyfunctional response of CD8+ memory T cells, and extending survival. Here, we show that combination therapy targets IDO signaling, specifically in MDSCs, tumor cells, and CD8+ T cells infiltrating the TME. Deficiency of IDO caused significant reduction in tumor burden, tumor-infiltrating MDSCs, GM-CSF, MDSC survival and infiltration of programmed death receptor-1 (PD-1)-expressing CD8+ T cells compared to controls. IDO−/− MDSCs downregulated nutrient-sensing AMP-activated protein kinase (AMPK) activity, but IDO−/− CD8+ T cells showed AMPK activation associated with enhanced effector function. Our studies provide proof-of-concept for the efficacy of this combination therapy in inhibiting IDO and T cell exhaustion in a syngeneic model of lung cancer and provide mechanistic insights for IDO-dependent metabolic reprogramming of MDSCs that reduces T cell exhaustion and regulates anti-tumor immunity. PMID:27705910

  15. Indoleamine 2,3-dioxygenase regulates anti-tumor immunity in lung cancer by metabolic reprogramming of immune cells in the tumor microenvironment.

    PubMed

    Schafer, Cara C; Wang, Yong; Hough, Kenneth P; Sawant, Anandi; Grant, Stefan C; Thannickal, Victor J; Zmijewski, Jaroslaw; Ponnazhagan, Selvarangan; Deshane, Jessy S

    2016-11-15

    Indoleamine 2,3-dioxygenase (IDO) has been implicated in immune evasion by tumors. Upregulation of this tryptophan (Trp)-catabolizing enzyme, in tumor cells and myeloid-derived suppressor cells (MDSCs) within the tumor microenvironment (TME), leads to Trp depletion that impairs cytotoxic T cell responses and survival; however, exact mechanisms remain incompletely understood. We previously reported that a combination therapy of gemcitabine and a superoxide dismutase mimetic promotes anti-tumor immunity in a mouse model of lung cancer by inhibiting MDSCs, enhancing polyfunctional response of CD8+ memory T cells, and extending survival. Here, we show that combination therapy targets IDO signaling, specifically in MDSCs, tumor cells, and CD8+ T cells infiltrating the TME. Deficiency of IDO caused significant reduction in tumor burden, tumor-infiltrating MDSCs, GM-CSF, MDSC survival and infiltration of programmed death receptor-1 (PD-1)-expressing CD8+ T cells compared to controls. IDO-/- MDSCs downregulated nutrient-sensing AMP-activated protein kinase (AMPK) activity, but IDO-/- CD8+ T cells showed AMPK activation associated with enhanced effector function. Our studies provide proof-of-concept for the efficacy of this combination therapy in inhibiting IDO and T cell exhaustion in a syngeneic model of lung cancer and provide mechanistic insights for IDO-dependent metabolic reprogramming of MDSCs that reduces T cell exhaustion and regulates anti-tumor immunity.

  16. Saturation Mutagenesis of Burkholderia cepacia R34 2,4-Dinitrotoluene Dioxygenase at DntAc Valine 350 for Synthesizing Nitrohydroquinone, Methylhydroquinone, and Methoxyhydroquinone

    PubMed Central

    Keenan, Brendan G.; Leungsakul, Thammajun; Smets, Barth F.; Wood, Thomas K.

    2004-01-01

    Saturation mutagenesis of the 2,4-dinitrotoluene dioxygenase (DDO) of Burkholderia cepacia R34 at position valine 350 of the DntAc α-subunit generated mutant V350F with significantly increased activity towards o-nitrophenol (47 times), m-nitrophenol (34 times), and o-methoxyphenol (174 times) as well as an expanded substrate range that now includes m-methoxyphenol, o-cresol, and m-cresol (wild-type DDO had no detectable activity for these substrates). Another mutant, V350M, also displays increased activity towards o-nitrophenol (20 times) and o-methoxyphenol (162 times) as well as novel activity towards o-cresol. Products were synthesized using whole Escherichia coli TG1 cells expressing the recombinant R34 dntA loci from pBS(Kan)R34, and the initial rates of product formation were determined at 1 mM substrate by reverse-phase high-pressure liquid chromatography. V350F produced both nitrohydroquinone at a rate of 0.75 ± 0.15 nmol/min/mg of protein and 3-nitrocatechol at a rate of 0.069 ± 0.001 nmol/min/mg of protein from o-nitrophenol, 4-nitrocatechol from m-nitrophenol at 0.29 ± 0.02 nmol/min/mg of protein, methoxyhydroquinone from o-methoxyphenol at 2.5 ± 0.6 nmol/min/mg of protein, methoxyhydroquinone from m-methoxyphenol at 0.55 ± 0.02 nmol/min/mg of protein, both methylhydroquinone at 1.52 ± 0.02 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.74 ± 0.05 nmol/min/mg of protein from o-cresol, and methylhydroquinone at 0.43 ± 0.1 nmol/min/mg of protein from m-cresol. V350M produced both nitrohydroquinone at a rate of 0.33 nmol/min/mg of protein and 3-nitrocatechol at 0.089 nmol/min/mg of protein from o-nitrophenol, methoxyhydroquinone from o-methoxyphenol at 2.4 nmol/min/mg of protein, methylhydroquinone at 1.97 nmol/min/mg of protein and 2-hydroxybenzyl alcohol at 0.11 nmol/min/mg of protein from o-cresol. The DDO variants V350F and V350M also exhibited 10-fold-enhanced activity towards naphthalene (8 ± 2.6 nmol/min/mg of protein), forming (1R

  17. The MarR-type repressor MhqR (YkvE) regulates multiple dioxygenases/glyoxalases and an azoreductase which confer resistance to 2-methylhydroquinone and catechol in Bacillus subtilis.

    PubMed

    Töwe, Stefanie; Leelakriangsak, Montira; Kobayashi, Kazuo; Van Duy, Nguyen; Hecker, Michael; Zuber, Peter; Antelmann, Haike

    2007-10-01

    Catechol and 2-methylhydroquinone (2-MHQ) cause the induction of the thiol-specific stress response and four dioxygenases/glyoxalases in Bacillus subtilis. Using transcription factor arrays, the MarR-type regulator YkvE was identified as a repressor of the dioxygenase/glyoxalase-encoding mhqE gene. Transcriptional and proteome analyses of the DeltaykvE mutant revealed the upregulation of ykcA (mhqA), ydfNOP (mhqNOP), yodED (mhqED) and yvaB (azoR2) encoding multiple dioxygenases/glyoxalases, oxidoreductases and an azoreductase. Primer extension experiments identified sigma(A)-type promoter sequences upstream of mhqA, mhqNOP, mhqED and azoR2 from which transcription is elevated after thiol stress. DNase I footprinting analysis showed that YkvE protects a primary imperfect inverted repeat with the consensus sequence of tATCTcgaAtTCgAGATaaaa in the azoR2, mhqE and mhqN promoter regions. Analysis of mhqE-promoter-bgaB fusions confirmed the significance of YkvE binding to this operator in vivo. Adjacent secondary repeats were protected by YkvE in the azoR2 and mhqN promoter regions consistent with multiple DNA-protein binding complexes. DNA-binding activity of YkvE was not directly affected by thiol-reactive compounds in vitro. Mutational analyses showed that MhqA, MhqO and AzoR2 confer resistance to 2-MHQ. Moreover, the DeltaykvE mutant displayed a 2-MHQ and catechol resistant phenotype. YkvE was renamed as MhqR controlling a 2-MHQ and catechol-resistance regulon of B. subtilis.

  18. Chronic lymphocytic leukemia nurse-like cells express hepatocyte growth factor receptor (c-MET) and indoleamine 2,3-dioxygenase and display features of immunosuppressive type 2 skewed macrophages

    PubMed Central

    Giannoni, Paolo; Pietra, Gabriella; Travaini, Giorgia; Quarto, Rodolfo; Shyti, Genti; Benelli, Roberto; Ottaggio, Laura; Mingari, Maria Cristina; Zupo, Simona; Cutrona, Giovanna; Pierri, Ivana; Balleari, Enrico; Pattarozzi, Alessandra; Calvaruso, Marco; Tripodo, Claudio; Ferrarini, Manlio; de Totero, Daniela

    2014-01-01

    Hepatocyte growth factor, produced by stromal and follicular dendritic cells, and present at high concentrations in the sera of patients with chronic lymphocytic leukemia, prolongs the survival of leukemic B cells by interacting with their receptor, c-MET. It is, however, unknown whether hepatocyte growth factor influences microenvironmental cells, such as nurse-like cells, which deliver survival signals to the leukemic clone. We evaluated the expression of c-MET on nurse-like cells and monocytes from patients with chronic lymphocytic leukemia and searched for phenotypic/functional features supposed to be influenced by the hepatocyte growth factor/c-MET interaction. c-MET is expressed at high levels on nurse-like cells and at significantly higher levels than normal on monocytes from patients. Moreover, the hepatocyte growth factor/c-MET interaction activates STAT3TYR705 phosphorylation in nurse-like cells. Indoleamine 2,3-dioxygenase, an enzyme modulating T-cell proliferation and induced on normal monocytes after hepatocyte growth factor treatment, was detected together with interleukin-10 on nurse-like cells, and on freshly-prepared patients’ monocytes. Immunohistochemical/immunostaining analyses demonstrated the presence of c-MET+ and indoleamine 2,3-dioxygenase+ cells in lymph node biopsies, co-expressed with CD68 and vimentin. Furthermore nurse-like cells and chronic lymphocytic monocytes significantly inhibited T-cell proliferation, prevented by anti-transforming growth factor beta and interleukin-10 antibodies and indoleamine 2,3-dioxygenase inhibitors, and supported CD4+CD25high+/FOXP3+ T regulatory cell expansion. We suggest that nurse-like cells display features of immunosuppressive type 2 macrophages: higher hepatocyte growth factor levels, produced by leukemic or other microenvironmental surrounding cells, may cooperate to induce M2 polarization. Hepatocyte growth factor may thus have a dual pathophysiological role: directly through enhancement of

  19. Expression of a putative dioxygenase gene adjacent to an insertion mutation is involved in the short internodes of columnar apples (Malus × domestica).

    PubMed

    Okada, Kazuma; Wada, Masato; Moriya, Shigeki; Katayose, Yuichi; Fujisawa, Hiroko; Wu, Jianzhong; Kanamori, Hiroyuki; Kurita, Kanako; Sasaki, Harumi; Fujii, Hiroshi; Terakami, Shingo; Iwanami, Hiroshi; Yamamoto, Toshiya; Abe, Kazuyuki

    2016-11-01

    Determining the molecular mechanism of fruit tree architecture is important for tree management and fruit production. An apple mutant 'McIntosh Wijcik', which was discovered as a bud mutation from 'McIntosh', exhibits a columnar growth phenotype that is controlled by a single dominant gene, Co. In this study, the mutation and the Co gene were analyzed. Fine mapping narrowed the Co region to a 101 kb region. Sequence analysis of the Co region and the original wild-type co region identified an insertion mutation of an 8202 bp long terminal repeat (LTR) retroposon in the Co region. Segregation analysis using a DNA marker based on the insertion polymorphism showed that the LTR retroposon was closely associated with the columnar growth phenotype. RNA-seq and RT-PCR analysis identified a promising Co candidate gene (91071-gene) within the Co region that is specifically expressed in 'McIntosh Wijcik' but not in 'McIntosh'. The 91071-gene was located approximately 16 kb downstream of the insertion mutation and is predicted to encode a 2-oxoglutarate-dependent dioxygenase involved in an unknown reaction. Overexpression of the 91071-gene in transgenic tobaccos and apples resulted in phenotypes with short internodes, like columnar apples. These data suggested that the 8202 bp retroposon insertion in 'McIntosh Wijcik' is associated with the short internodes of the columnar growth phenotype via upregulated expression of the adjacent 91071-gene. Furthermore, the DNA marker based on the insertion polymorphism could be useful for the marker-assisted selection of columnar apples.

  20. An active-site phenylalanine directs substrate binding and C-H cleavage in the alpha-ketoglutarate-dependent dioxygenase TauD.

    PubMed

    McCusker, Kevin P; Klinman, Judith P

    2010-04-14

    Enzymes that cleave C-H bonds are often found to depend on well-packed hydrophobic cores that influence the distance between the hydrogen donor and acceptor. Residue F159 in taurine alpha-ketoglutarate dioxygenase (TauD) is demonstrated to play an important role in the binding and orientation of its substrate, which undergoes a hydrogen atom transfer to the active site Fe(IV)=O. Mutation of F159 to smaller hydrophobic side chains (L, V, A) leads to substantially reduced rates for substrate binding and for C-H bond cleavage, as well as increased contribution of the chemical step to k(cat) under steady-state turnover conditions. The greater sensitivity of these substrate-dependent processes to mutation at position 159 than observed for the oxygen activation process supports a previous conclusion of modularity of function within the active site of TauD (McCusker, K. P.; Klinman, J. P. Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 19791-19795). Extraction of intrinsic deuterium kinetic isotope effects (KIEs) using single turnover transients shows 2- to 4-fold increase in the size of the KIE for F159V in relation to wild-type and F159L. It appears that there is a break in behavior following removal of a single methylene from the side chain of F159L to generate F159V, whereby the protein active site loses its ability to restore the internuclear distance between substrate and Fe(IV)=O that supports optimal hydrogenic wave function overlap.

  1. Robust Inhibitory Effects of Conjugated Linolenic Acids on a Cyclooxygenase-related Linoleate 10S-Dioxygenase: Comparison with COX-1 and COX-2

    PubMed Central

    Mashhadi, Zahra; Boeglin, William E.; Brash, Alan R.

    2015-01-01

    There are many reports of the anti-inflammatory, anti-cancer, and anti-atherosclerotic activities of conjugated linolenic acids (cLNA). They constitute a small percentage of fatty acids in the typical human diet, although up to 80% of the fatty acids in certain fruits such as pomegranate. In the course of studying a bacterial fatty acid dioxygenase (Nostoc linoleate 10S-DOX, an ancient relative of mammalian cyclooxygenases), we detected strong inhibitory activity in a commercial sample of linoleic acid. We identified two cLNA isomers, β-eleostearic (9E,11E,13E-18:3) and β-calendic acid (8E,10E,12E-18:3), as responsible for that striking inhibition with a Ki of ~49 nM and ~125 nM, respectively, the most potent among eight cLNA tested. We also examined the effects of all eight cLNA on the activity of COX-1 and COX-2. Jacaric acid (8Z,10E,12Z-18:3) and its 12E isomer, 8Z,10E,12E-18:3, strongly inhibit the activity of COX-1 with a Ki of ~1.7 and ~1.1 μM, respectively. By contrast, COX-2 was ≤ 30% inhibited at 10μM concentrations of the cLNA. Identifying the activities of the naturally occurring fatty acids is of interest in terms of understanding their interaction with the enzymes, and for explaining the mechanistic basis of their biological effects. The study also highlights the potential presence of inhibitory fatty acids in commercial lipids prepared from natural sources. Analysis of seven commercial samples of linoleic acid by HPLC and UV spectroscopy is illustrated as supplementary data. PMID:26209563

  2. Expression and regulation of immune-modulatory enzyme indoleamine 2,3-dioxygenase (IDO) by human airway epithelial cells and its effect on T cell activation

    PubMed Central

    Sewell, Herb F.; Knox, Alan; Ghaemmaghami, Amir M.

    2016-01-01

    Indoleamine 2,3-dioxygenase (IDO) catalyzes the degradation of tryptophan, which plays a critical role in immune suppression through regulating the production of a series of metabolites that are generally referred to as kynurenines. It has become increasingly clear that epithelial cells (ECs) play an active role in maintaining lung homeostasis by modulating the function of immune cells via producing cytokines, chemokines, and anti-microbial mediators. In this study we assessed the regulation of IDO activity and expression in human primary ECs and EC lines under steady state conditions and in response to bacterial and allergenic stimuli. We also investigated the potential immune modulatory functions of IDO expression in human airway ECs. Our data clearly show that airway ECs produce IDO, which is down-regulated in response to allergens and TLR ligands while up-regulated in response to IFN-γ. Using gene silencing, we further demonstrate that IDO plays a key role in the EC-mediated suppression of antigen-specific and polyclonal proliferation of T cells. Interestingly, our data also show that ECs lose their inhibitory effect on T cell activation in response to different TLR agonists mimicking bacterial or viral infections. In conclusion, our work provides an understanding of how IDO is regulated in ECs as well as demonstrates that “resting” ECs can suppress T cell activation in an IDO dependent manner. These data provide new insight into how ECs, through the production of IDO, can influence downstream innate and adaptive responses as part of their function in maintaining immune homeostasis in the airways. PMID:27613847

  3. Addition of an Indoleamine-2,3,-dioxygenase Inhibitor to B cell Depletion Therapy Blocks Autoreactive B Cell Activation and Recurrence of Arthritis

    PubMed Central

    Pigott, Elizabeth; Mandik-Nayak, Laura

    2012-01-01

    Objective Define the role indoleamine-2,3-dioxygenase (IDO) plays in driving pathogenic B cells responses leading to arthritis and determine if inhibitors of the IDO pathway can be used in conjunction with B cell depletion therapy to prevent the re-emergence of autoantibodies and arthritis following reconstitution of the B cell repertoire. Methods Immunoglobulin transgenic mice were treated with the IDO inhibitor 1-methyl-tryptophan (1MT) and followed for the extent of autoreactive B cell activation. Arthritic mice (K/BxN) were treated with B cell depletion therapy alone or in combination with 1MT. Mice were followed for the presence of autoantibody secreting cells, inflammatory cytokines, and joint inflammation. Results 1MT did not affect the initial activation or survival of autoreactive B cells, but did inhibit their ability to differentiate into autoantibody secreting cells. Treatment with anti-CD20 depleted the B cell repertoire and attenuated arthritis symptoms; however, arthritis symptoms rapidly returned as B cells repopulated the repertoire. Administration of 1MT prior to B cell repopulation prevented the production of autoantibodies, inflammatory cytokines, and flare in arthritis symptoms. Conclusion IDO activity is essential for the differentiation of autoreactive B cells into antibody secreting cells, but is not necessary for their initial stages of activation. Addition of 1MT to B cell depletion therapy prevents the differentiation of autoantibody secreting cells and recurrence of autoimmune arthritis following reconstitution of the B cell repertoire. These data suggest that IDO inhibitors could be used in conjunction with B cell depletion as an effective co-therapeutic strategy in the treatment of rheumatoid arthritis. PMID:22294267

  4. Impact of immunosuppressive agents on the expression of indoleamine 2,3-dioxygenase, heme oxygenase-1 and interleukin-7 in mesangial cells.

    PubMed

    Liang, Guo-Biao; Luo, Guang-Heng; Bao, Ding-Su; Chen, An-Jian; Zhuang, Yong-Xiang; Guo, Ya-Nan; Wang, Xin; Wang, Yuan-Liang; Chen, Zong-Ping; Lu, Yi-Ping; Li, You-Ping

    2015-08-01

    Chronic allograft nephropathy (CAN) is a major cause of graft loss following kidney transplantation and may result from the interactions of various immune and non-immune factors. The aim of the present study was to establish an in vitro model of glomerular mesangial cell injury in order to examine the gene expression levels of indoleamine 2,3-dioxygenase (IDO), heme oxygenase-1 (HO-1) and interleukin-7 (IL-7) in mesangial cells during the healing process as well as to investigate the effects of various immunosuppressants on the expression of these genes. The HBZY-1 glomerular mesangial cell line was pre-treated in vitro with cytochalasin B for 2 h to induce reversible damage. Following the pre-treatment, the HBZY-1 cells were divided into five groups: Blank control group, cyclosporine A (CsA) group, tacrolimus (Tac) group, mycophenolate mofetil (MMF) group and rapamycin (RAPA) group. After treating the mesangial cells with each immunosuppressive drug for 6, 12 or 24 h, the mRNA and protein expression levels of IDO, HO-1 and IL-7 were examined using reverse transcription quantitative polymerase chain reaction (RT-qPCR), western blot and immunohistochemical analyses. The results showed that expression levels of HO-1 were significantly upregulated in response to treatment with CsA, FK506, RAPA and MMF, whereas the expression levels of IL-7 were markedly downregulated by treatment with the above immunosuppressants. CsA, FK506 and MMF significantly enhanced the expression levels of IDO, whereas RAPA exhibited no apparent effect on IDO. The present study may contribute to the understanding of the pathogenesis of CAN and provide novel strategies for the prevention and treatment of CAN.

  5. Effect of overexpression of citrus 9-cis-epoxycarotenoid dioxygenase 3 (CsNCED3) on the physiological response to drought stress in transgenic tobacco.

    PubMed

    Pedrosa, A M; Cidade, L C; Martins, C P S; Macedo, A F; Neves, D M; Gomes, F P; Floh, E I S; Costa, M G C

    2017-03-30

    9-cis-epoxycarotenoid dioxygenase (NCED) encodes a key enzyme in abscisic acid (ABA) biosynthesis. Little is known regarding the regulation of stress response by NCEDs at physiological levels. In the present study, we generated transgenic tobacco overexpressing an NCED3 ortholog from citrus (CsNCED3) and investigated its relevance in the regulation of drought stress tolerance. Wild-type (WT) and transgenic plants were grown under greenhouse conditions and subjected to drought stress for 10 days. Leaf predawn water potential (Ψwleaf), stomatal conductance (gs), net photosynthetic rate (A), transpiration rate (E), instantaneous (A/E) and intrinsic (A/gs) water use efficiency (WUE), and in situ hydrogen peroxide (H2O2) and abscisic acid (ABA) production were determined in leaves of irrigated and drought-stressed plants. The Ψwleaf decreased throughout the drought stress period in both WT and transgenic plants, but was restored after re-watering. No significant differences were observed in gs between WT and transgenic plants under normal conditions. However, the transgenic plants showed a decreased (P ≤ 0.01) gs on the 4th day of drought stress, which remained lower (P ≤ 0.001) than the WT until the end of the drought stress. The A and E levels in the transgenic plants were similar to those in WT; therefore, they exhibited increased A/gs under drought conditions. No significant differences in A, E, and gs values were observed between the WT and transgenic plants after re-watering. The transgenic plants had lower H2O2 and higher ABA than the WT under drought conditions. Our results support the involvement of CsNCED3 in drought avoidance.

  6. Robust inhibitory effects of conjugated linolenic acids on a cyclooxygenase-related linoleate 10S-dioxygenase: Comparison with COX-1 and COX-2.

    PubMed

    Mashhadi, Zahra; Boeglin, William E; Brash, Alan R

    2015-10-01

    There are many reports of the anti-inflammatory, anti-cancer, and anti-atherosclerotic activities of conjugated linolenic acids (cLNA). They constitute a small percentage of fatty acids in the typical human diet, although up to 80% of the fatty acids in certain fruits such as pomegranate. In the course of studying a bacterial fatty acid dioxygenase (Nostoc linoleate 10S-DOX, an ancient relative of mammalian cyclooxygenases), we detected strong inhibitory activity in a commercial sample of linoleic acid. We identified two cLNA isomers, β-eleostearic (9E,11E,13E-18:3) and β-calendic acid (8E,10E,12E-18:3), as responsible for that striking inhibition with a Ki of ~49nM and ~125nM, respectively, the most potent among eight cLNA tested. We also examined the effects of all eight cLNA on the activity of COX-1 and COX-2. Jacaric acid (8Z,10E,12Z-18:3) and its 12E isomer, 8Z,10E,12E-18:3, strongly inhibit the activity of COX-1 with a Ki of ~1.7 and ~1.1μM, respectively. By contrast, COX-2 was ≤30% inhibited at 10μM concentrations of the cLNA. Identifying the activities of the naturally occurring fatty acids is of interest in terms of understanding their interaction with the enzymes, and for explaining the mechanistic basis of their biological effects. The study also highlights the potential presence of inhibitory fatty acids in commercial lipids prepared from natural sources. Analysis of seven commercial samples of linoleic acid by HPLC and UV spectroscopy is illustrated as supplementary data.

  7. Tryptophan-2,3-dioxygenase is regulated by prostaglandin E2 in malignant glioma via a positive signaling loop involving prostaglandin E receptor-4.

    PubMed

    Ochs, Katharina; Ott, Martina; Rauschenbach, Katharina J; Deumelandt, Katrin; Sahm, Felix; Opitz, Christiane A; von Deimling, Andreas; Wick, Wolfgang; Platten, Michael

    2015-12-27

    Malignant gliomas and other types of tumors generate a local immunosuppressive microenvironment, which prohibits an effective anti-tumor immune response and promotes tumor growth. Along with others, we have recently demonstrated that catabolism of the essential amino acid tryptophan via tryptophan-2,3-dioxygenase (TDO) is an important mechanism mediating tumor-associated immunosuppression particularly in gliomas. The pathways regulating TDO in tumors, however, are poorly understood. Here we show that prostaglandins enhance TDO expression and enzymatic activity in malignant gliomas via activation of prostaglandin E receptor-4 (EP4). Stimulation with prostaglandin E2 (PGE2 ) up-regulated TDO-mediated kynurenine release in human glioma cell lines while knockdown of the PGE2 receptor EP4 inhibited TDO expression and activity. In human malignant glioma tissue expression of the PGE2 -producing enzyme cyclooxygenase-2 (COX2) and its receptor EP4 were associated with TDO expression both on transcript and protein level. High expression of EP4 correlated with poor survival in malignant glioma patients WHO III-IV. Importantly, treatment of glioma cells with an EP4 inhibitor decreased TDO expression and activity. Moreover, TDO-over-expressing murine gliomas showed increased COX2 and EP4 expression suggesting a positive feedback mechanism in vivo. In summary, targeting EP4 may inhibit - in addition to immunosuppressive COX2 signaling - tryptophan degradation as another important immunosuppressive pathway and thus, could provide a dual clinically relevant immunotherapeutic avenue for the treatment of malignant gliomas. This article is protected by copyright. All rights reserved.

  8. Mouse chronic social stress increases blood and brain kynurenine pathway activity and fear behaviour: Both effects are reversed by inhibition of indoleamine 2,3-dioxygenase.

    PubMed

    Fuertig, René; Azzinnari, Damiano; Bergamini, Giorgio; Cathomas, Flurin; Sigrist, Hannes; Seifritz, Erich; Vavassori, Stefano; Luippold, Andreas; Hengerer, Bastian; Ceci, Angelo; Pryce, Christopher R

    2016-05-01

    Psychosocial stress is a major risk factor for mood and anxiety disorders, in which excessive reactivity to aversive events/stimuli is a major psychopathology. In terms of pathophysiology, immune-inflammation is an important candidate, including high blood and brain levels of metabolites belonging to the kynurenine pathway. Animal models are needed to study causality between psychosocial stress, immune-inflammation and hyper-reactivity to aversive stimuli. The present mouse study investigated effects of psychosocial stress as chronic social defeat (CSD) versus control-handling (CON) on: Pavlovian tone-shock fear conditioning, activation of the kynurenine pathway, and efficacy of a specific inhibitor (IDOInh) of the tryptophan-kynurenine catabolising enzyme indoleamine 2,3-dioxygenase (IDO1), in reversing CSD effects on the kynurenine pathway and fear. CSD led to excessive fear learning and memory, whilst repeated oral escitalopram (antidepressant and anxiolytic) reversed excessive fear memory, indicating predictive validity of the model. CSD led to higher blood levels of TNF-α, IFN-γ, kynurenine (KYN), 3-hydroxykynurenine (3-HK) and kynurenic acid, and higher KYN and 3-HK in amygdala and hippocampus. CSD was without effect on IDO1 gene or protein expression in spleen, ileum and liver, whilst increasing liver TDO2 gene expression. Nonetheless, oral IDOInh reduced blood and brain levels of KYN and 3-HK in CSD mice to CON levels, and we therefore infer that CSD increases IDO1 activity by increasing its post-translational activation. Furthermore, repeated oral IDOInh reversed excessive fear memory in CSD mice to CON levels. IDOInh reversal of CSD-induced hyper-activity in the kynurenine pathway and fear system contributes significantly to the evidence for a causal pathway between psychosocial stress, immune-inflammation and the excessive fearfulness that is a major psychopathology in stress-related neuropsychiatric disorders.

  9. Homogentisate 1-2-Dioxygenase Downregulation in the Chronic Persistence of Pseudomonas aeruginosa Australian Epidemic Strain-1 in the CF Lung.

    PubMed

    Harmer, Christopher J; Wynn, Matthew; Pinto, Rachel; Cordwell, Stuart; Rose, Barbara R; Harbour, Colin; Triccas, James A; Manos, Jim

    2015-01-01

    Some Pseudomonas aeruginosa strains including Australian Epidemic Strain-1 (AES-1 or AUS-01) cause persistent chronic infection in cystic fibrosis (CF) patients, with greater morbidity and mortality. Factors conferring persistence are largely unknown. Previously we analysed the transcriptomes of AES-1 grown in Luria broth, nematode growth medium for Caenorhabditis elegans assay (both aerobic) and artificial sputum medium (mainly hypoxic). Transcriptional comparisons included chronic AES-1 strains against PAO1 and acute AES-1 (AES-1R) against its chronic isogen (AES-1M), isolated 10.5 years apart from a CF patient and not eradicated in the meantime. Prominent amongst genes downregulated in AES-1M in all comparisons was homogentisate-1-2-dioxygenase (hmgA); an oxygen-dependent gene known to be mutationally deactivated in many chronic infection strains of P. aeruginosa. To investigate if hmgA downregulation and deactivation gave similar virulence persistence profiles, a hmgA mutant made in UCBPP-PA14 utilising RedS-recombinase and AES-1M were assessed in the C. elegans virulence assay, and the C57BL/6 mouse for pulmonary colonisation and TNF-α response. In C. elegans, hmgA deactivation resulted in significantly increased PA14 virulence while hmgA downregulation reduced AES-1M virulence. AES-1M was significantly more persistent in mouse lung and showed a significant increase in TNF-α (p<0.0001), sustained even with no detectable bacteria. PA14ΔhmgA did not show increased TNF-α. This study suggests that hmgA may have a role in P. aeruginosa persistence in chronic infection and the results provide a starting point for clarifying the role of hmgA in chronic AES-1.

  10. Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation.

    PubMed

    Zhang, Yuan; Wang, Ting; Yang, Ke; Xu, Ji; Ren, Lijie; Li, Weiping; Liu, Wenlan

    2016-01-01

    Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1's downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke.

  11. Cerebral Microvascular Endothelial Cell Apoptosis after Ischemia: Role of Enolase-Phosphatase 1 Activation and Aci-Reductone Dioxygenase 1 Translocation

    PubMed Central

    Zhang, Yuan; Wang, Ting; Yang, Ke; Xu, Ji; Ren, Lijie; Li, Weiping; Liu, Wenlan

    2016-01-01

    Enolase-phosphatase 1 (ENOPH1), a newly discovered enzyme of the methionine salvage pathway, is emerging as an important molecule regulating stress responses. In this study, we investigated the role of ENOPH1 in blood brain barrier (BBB) injury under ischemic conditions. Focal cerebral ischemia induced ENOPH1 mRNA and protein expression in ischemic hemispheric microvessels in rats. Exposure of cultured brain microvascular endothelial cells (bEND3 cells) to oxygen-glucose deprivation (OGD) also induced ENOPH1 upregulation, which was accompanied by increased cell death and apoptosis reflected by increased 3-(4, 5-Dimethylthiazol-2-yl)-2, 5- diphenyltetrazolium bromide formation, lactate dehydrogenase release and TUNEL staining. Knockdown of ENOPH1 expression with siRNA or overexpressing ENOPH1 with CRISPR-activated plasmids attenuated or potentiated OGD-induced endothelial cell death, respectively. Moreover, ENOPH1 knockdown or overexpression resulted in a significant reduction or augmentation of reactive oxygen species (ROS) generation, apoptosis-associated proteins (caspase-3, PARP, Bcl-2 and Bax) and Endoplasmic reticulum (ER) stress proteins (Ire-1, Calnexin, GRP78 and PERK) in OGD-treated endothelial cells. OGD upregulated the expression of ENOPH1’s downstream protein aci-reductone dioxygenase 1 (ADI1) and enhanced its interaction with ENOPH1. Interestingly, knockdown of ENOPH1 had no effect on OGD-induced ADI1 upregulation, while it potentiated OGD-induced ADI1 translocation from the nucleus to the cytoplasm. Lastly, knockdown of ENOPH1 significantly reduced OGD-induced endothelial monolayer permeability increase. In conclusion, our data demonstrate that ENOPH1 activation may contribute to OGD-induced endothelial cell death and BBB disruption through promoting ROS generation and the activation of apoptosis associated proteins, thus representing a new therapeutic target for ischemic stroke. PMID:27630541

  12. Comparative study of putative 9-cis-epoxycarotenoid dioxygenase and abscisic acid accumulation in the responses of Sunki mandarin and Rangpur lime to water deficit.

    PubMed

    Neves, D M; Filho, M A Coelho; Bellete, B S; Silva, M F G F; Souza, D T; Dos S Soares Filho, W; Costa, M G C; Gesteira, A S

    2013-09-01

    Abscisic acid is a plant hormone that participates in essential plant physiological processes, especially during adaptation to many environmental stresses, such as water deficit. The relationship between ABA accumulation and the expression of putative carotenoid cleavage dioxygenase (CCD) genes was investigated in the pot-cultivated leaves and roots of the 'Rangpur' lime and 'Sunki Maravilha' mandarin plants. Transpiration, stomatal resistance and leaf growth were evaluated when these genotypes were subjected to continuous water deficit. Under water deficit conditions, the 'Rangpur' lime extracts used greater amounts of water when compared to the 'Sunki Maravilha' plants, which reached the greatest stomatal resistance 5 days before 'Rangpur' lime. When subjected to water deficit, the roots and leaves of 'Sunki Maravilha' showed a progressive increase in ABA accumulation; however, in 'Rangpur' lime, alternations between high and low ABA concentrations were observed. These results suggest a retroactive feeding regulation by ABA. In 'Rangpur' lime the NCED2, NCED3 and CCD4a genes were expressed at the highest levels in the roots, and NCED5 was highly expressed in the leaves; in 'Sunki Maravilha', the NCED2 and NCED5 genes were most highly expressed in the roots, and NCED2 was most highly expressed in the leaves. However, for both genotypes, the transcription of these genes only correlated with ABA accumulation during the most severe water deficit conditions. The 'Rangpur' lime behaved as a vigorous rootstock; the leaf growth remained unaltered even when water was scarce. However, 'Sunki Maravilha' adaptation was based on the equilibrium of the response between the root and the aerial tissues due to water restriction. The use of the Sunki mandarin in combination with a scion with similar characteristics as its own, which responds to water deficit stress by accumulating ABA in the leaves, may display good drought tolerance under field conditions.

  13. The Synergistic Local Immunosuppressive Effects of Neural Stem Cells Expressing Indoleamine 2,3-Dioxygenase (IDO) in an Experimental Autoimmune Encephalomyelitis (EAE) Animal Model.

    PubMed

    Lee, Young Eun; An, Jaeyeol; Lee, Kee-Hang; Kim, Sung Su; Song, Hye Jin; Pyeon, Heejang; Nam, Hyun; Kang, Kyeongjin; Joo, Kyeung Min

    2015-01-01

    Neurodegenerative diseases provoke robust immunological reactions in the central nervous system (CNS), which further deteriorate the neural tissue damage. We hypothesized that the expression levels of indoleamine 2,3-dioxygenase (IDO), an enzyme that has potent immune suppressive activities, in neural stem cells (NSCs) would have synergistic therapeutic effects against neurodegenerative diseases, since NSCs themselves have low IDO expression. In this study, the synergistic immune suppressive effects of rat fetal NSCs expressing IDO (rfNSCs-IDO) were validated by mixed leukocyte reaction (MLR) in vitro and an experimental autoimmune encephalomyelitis (EAE) animal model in vivo. rfNSCs-IDO showed significantly more suppressive effects on T cell proliferation in the MLR compared to control rfNSCs (rfNSCs-Cont). Importantly, IDO inhibition using 1-methyl-DL-tryptophan (1-MT), an IDO inhibitor, reversed the synergistic effects, confirming IDO-specific effects in rfNSCs-IDO. In the EAE animal model, systemic rfNSCs-IDO injections resulted in significant local immune suppression in the cervical lymph nodes and CNS, evidenced by a reduction in the number of activated T lymphocytes and an increase in regulatory T cell numbers, which induced significantly fewer clinical symptoms and faster recovery. In contrast, rfNSCs-Cont failed to reduce symptoms in the EAE animal models, although they showed local immune suppression, which was significantly less than that in rfNSCs-IDO. Taken together, IDO expression in NSCs synergistically potentiates the immune suppression activities of NSCs and could be applicable for the development of therapeutic modalities against various neurodegenerative diseases.

  14. Regulated nucleo-cytoplasmic shuttling of human aci-reductone dioxygenase (hADI1) and its potential role in mRNA processing.

    PubMed

    Gotoh, Isamu; Uekita, Takamasa; Seiki, Motoharu

    2007-01-01

    Bacterial aci-reductone dioxygenase (ARD), a member of the cupin superfamily, has evolutionarily primitive protein folding and functions in the methionine recycling pathway. Recently, a human ARD orthologue (human ADI1, hADI1) has been identified and exhibits functions other than ARD activity. The hADI1 localizes mainly to the cytoplasm, but a substantial fraction is nuclear, suggesting functions in both cellular compartments. In this study, we report that nucleo-cytoplasmic transport of hADI1 is regulated by a non-canonical nuclear export signal (NES) located in the N-terminal region of hADI1. The NES is composed of multiple basic amino-acid residues instead of the canonical leucine-rich sequence. Nuclear export of hADI1 was not mediated by CRM1, a major transporter that binds to leucine-rich NES. Substitution of the basic residues with alanines abolished NES activity. Mutant hADI1 accumulated in the nucleus and formed speckles frequently observed with splicing factors and some transcription factors. Indeed, hADI1 specifically co-localized with the splicing factor U1-70K to the nucleus but not with another splicing factor, SC35. U1-70K over-expression induced nuclear accumulation of hADI1. Nuclear hADI1 expression significantly altered the splicing pattern of the adenovirus E1A mini-gene, which generates multiple alternatively spliced transcripts. Thus, hADI1 may have acquired a novel role in nuclear mRNA processing possibly by modulating U1-70K-related functions, an activity negatively regulated by a non-classical NES sequence.