Anthranilate Hydroxylase from Aspergillus niger: Evidence for the Participation of Iron in the Double Hydroxylation Reaction

Evidence was obtained for the participation of iron in the double hydroxylation reaction catalyzed by anthranilate hydroxylase from Aspergillus niger (UBC 814). Omission of iron from the growth medium gave inactive preparations of anthranilate hydroxylase which could be reactivated by incubating the enzyme preparations with ferric citrate. The enzyme was susceptible to inhibition by metal chelating agents. The Ki for o-phenanthroline, which inhibited the enzyme activity non-competitively with respect to anthranilate, was calculated to be 0.9 mM. The inhibition by o-phenanthroline was counteracted by ferric complexes such as ferric-ethylenediaminetetraacetic acid and ferric citrate. Anthranilate afforded protection against inhibition by o-phenanthroline.

2,3-Dihydroxybenzoate 2,3-oxygenase from the chloroplast fraction of Tecoma stans

2,3-Dihydroxybenzoate-2,3-oxygenase is mainly localized in the soluble and the chloroplast fractions of Tecoma leaves. It is associated with the lamellar structure of the chloroplast fraction. The chloroplast enzyme has properties similar to those of the soluble enzyme, but it has a longer half-life and is more stable to dialysis than the soluble enzyme. It is inhibited by sulfhydryl reagents and the inhibition is reversed by the addition of reduced glutathione. The chloroplast enzyme is insensitive to iron-chelating agents. The enzyme loses activity on dialysis against copper-chelating agents and the activity is completely recovered on the addition of copper; addition of iron does not restore the activity. Polyphenol oxidase is probably present only in the active form in the Tecoma chloroplast but it is not involved in the intradiol cleavage of 2,3-dihydroxybenzoic acid.

A new mode of ring cleavage of 2,3-dihydroxybenzoic acid in Tecoma stans (L.). Partial purification and properties of 2,3-dihydroxybenzoate 2,3-oxygenase.

2,3-Dihydroxybenzoic acid has been shown to be oxidized via the 3-oxoadipate pathway in the leaves of Tecoma stans. The formation of 2-carboxy-cis,cis-muconic acid, a muconolactone, 3-oxoadipic acid and carbon dioxide during its metabolism has been demonstrated using an extract of Tecoma leaves. The first reaction of the pathway, viz., the conversion of 2,3-dihydroxybenzoate to 2-carboxy-cis,cis-muconic acid has been shown to be catalysed by an enzyme designated as 2,3-dihydroxybenzoate 2,3-oxygenase. The enzyme has been partially purified and a few of its properties studied. The enzyme is very labile with a half-life of 3--4 h. It is maximally active with 2,3-dihydroxybenzoate as the substrate and does not exhibit any activity with catechol, 4-methyl catechol, 3,4-dihydroxybenzoic acid, etc. However, 2,3-dihydroxy-p-toluate and 2,3-dihydroxy-p-cumate are also oxidized by the enzyme by about 38% and 28% respectively, compared to 2,3-dihydroxybenzoate. Sulfhydryl reagents inhibit the enzyme reaction and the inhibition can be prevented by preincubation of the enzyme with the substrate. Substrate also affords protection to the enzyme against thermal inactivation. Sulfhydryl compounds strongly inhibit the reaction and the inhibition cannot be prevented by preincubation of the enzyme with its substrates. Data on the effect of metal ions as well as metal chelating agents suggest that copper is the metal cofactor of the enzyme. Evidence is presented which suggests that iron may not be participating in the overall catalytic mechanism.

Purification and Properties of l-4-Hydroxymandelate Oxidase from Pseudomonas convexa

An inducible membrane-bound l-4-hydroxymandelate oxidase (decarboxylating) from Pseudomonas convexa has been solubilized and partially purified. It catalyzes the conversion of l-4-hydroxymandelic acid to 4-hydroxybenzaldehyde in a single step with the stoichiometric consumption of O2 and liberation of CO2. The enzyme is optimally active at pH 6.6 and at 55 oC. It requires FAD and Mn2+ for its activity. The membrane-bound enzyme is more stable than the solubilized and purified enzyme. After solubilization it gradually loses its activity when kept at 5 oC which can be fully reactivated by freezing and thawing. The Km values for DL-4-hydroxymandelate and FAD are 0.44 mM and 0.038 mM respectively. The enzyme is highly specific for DL-4-hydroxymandelic acid. DL-3,4-Dihydroxymandelic acid competitively inhibited the enzyme reaction. From the Dixon plot the Ki for DL-3,4-dihydroxymandelic acid was calculated to be 1.8 × 10−4 M. The enzyme is completely inactivated by thiol compounds and not affected by thiol inhibitors. The enzyme is also inhibited by denaturing agents, heavy metal ions and by chelating agents.

Comparison of partial extraction reagents for assessing potential bioavailability of heavy metals in sediments

Assessment of heavy metal bioavailability in sediments is complex because of the number of partial extraction methods available for the assessment and the general lack of certified reference materials. This study evaluates five different extraction methodologies to ascertain the relative strengths and weaknesses of each method. The results are then compared to previously published work to ascertain the most effective partial extraction technique, which was established to be dilute (0.75 – 1 M) nitric acid solutions. These results imply that single reagent; weak acid extractions provide a better assessment of potentially bioavailable metals than the chelating agents used in sequential extraction methods.

Indoleacetaldoxime hydro-lyase : II. Purification and properties

The purification and some properties of the enzyme indoleacetaldoxime hydrolyase (EC 4.2.1.29) from the fungus Gibberella fujikuroi, which dehydrates indoleacetaldoxime (IAOX) to indoleacetonitrile (IAN), are described. The enzyme activity in the fungus is present only under certain culture conditions. It is a soluble enzyme, has an optimum pH at 7, shows an energy of activation of —15,670 cal/mole, and has a Michaelis constant of 1.7 × 10−4 Image at 30 °. It appears to be specific for IAOX, and 1 mole of IAN is produced per mole of IAOX utilized. The enzyme is inhibited by a number of aldoximes of which phenylacetaldoxime (PAOX) is the most potent inhibitor. Inhibition by PAOX is competitive (Ki = 2.2 × 10−8 Image ). The enzyme is inhibited by SH reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide, and by a number of SH compounds such as cysteine, β-mercaptoethanol, and 2,3-dimercaptopropanol (BAL). However, glutathione activates the enzyme. Metal chelating agents such as 8-OH-quinoline and diethyl dithiocarbamate inhibit the enzyme; the inhibition is partly reversed by ferric citrate. Ascorbic acid, and particularly dehydroascorbic acid (DHA), are good activators of the enzyme. Several other biological oxidants had either no action or had a slight effect. Potassium cyanide activates the enzyme at low concentration but inhibits at higher concentrations. Reduction of the enzyme with NaBH4 reduces activity, and the effect is partly reversed by pyridoxal phosphate and also by DHA. The above properties indicate that both an SH function and an oxidized function are required for activity.

Studies on the metabolism of o-aminophenol purification and properties of isophenoxazine synthase from Bauhenia monandra

An enzyme which catalyzes the oxidative conversion of o-aminophenol to 2-amino-3-H-isophenoxazin-3-one has been purified 396-fold by using standard fractionation procedures. The enzyme is specific for o-aminophenol and has pH and temperature optima at 6.2 and 40 °, respectively. It is insensitive to metal chelating agents but is inhibited by several reducing substances. There is no cofactor or metal ion requirement for the reaction. A competitive type of inhibition was observed with structural analogs such as anthranilic acid and 3-hydroxyanthranilic acid. There are no free sulfhydryl groups in the enzyme, but preincubation of the enzyme with substrate or substrate analogs resulted in the liberation of titratable free sulfhydryl groups. The mechanism of biosynthesis of isophenoxazine ring is discussed.

Anthranilic acid oxidase system of Tecomastans—III.: Studies on the conversion of o-aminophenol to catechol

The terminal step in the oxidation of anthranilic acid to catechol by anthranilic acid oxidase system from Tecoma stans, which converts o-aminophenol to catechol has been studied in detail. The reaction catalyses the conversion of one molecule of o-aminophenol to one molecule each of ammonia and catechol. The partially purified enzyme has a pH optimum of 6·2 in citrate-phosphate buffer and a temperature optimum of 45°. The metal ions, Mg2+, Co2+ and Fe3+ were inhibitory to the reaction. Metal chelating agents like 8-hydroxyquinoline, o-phenanthroline, and diethyldithiocarbamate, caused a high degree of inhibition. A sulfhydryl requirement for the reaction was inferred from the inhibition of the reaction by p-chloromercuribenzoate and its reversal with GSH. Atebrin inhibition was reversed by addition of FAD to the reaction mixture.

Conversion of isophenoxazine to catechol in Tecoma stans

Isophenoxazine, formed by the condensation of two molecules of o-aminophenol, is reduced by an enzyme system from Tecoma stans leaves to two molecules of catechol. The reaction proceeds well under anaerobic conditions; a 1–2 mole stoichiometry between the substrate disappeared and the product formed was maintained. The enzyme showed maximum activity at pH 5. The substrate at high concentrations caused a diminution in the activity and the optimum concentration of substrate was at 6 × 10−4 Image . The enzyme preparation was able to convert cinnabarinic acid and diphenylene dioxide 2,3-quinone into the corresponding catechol substances. The diphenylene dioxide 2,3-quinone at the same concentration was three times more susceptible to enzymic cleavage than isophenoxazine. Cinnabarinic acid inhibited the enzymic cleavage of isophenoxazine competitively. None of the known electron donors was found to activate the reaction. Inhibition studies suggested that intact sulfhydryl groups are necessary for enzyme activity. Heavy metal ions like Hg++, Ag+, Co++, Fe++, Ni++, and Fe3++ inhibited the reaction. Metal chelating agents did not have any effect on the enzyme.

Anthranilic acid oxidase system of Tecoma stans—II. : Studies on the properties of a purified anthranilic acid oxidase system and its separation into different enzymic components

An enzyme system which catalysed the conversion of anthranilic acid to catechol has been purified 20-fold from a cell-free leaf extract of Tecoma stans. The optimum substrate concentration was 10−3 M and optimum temperature for the reaction was 45°. The presence of a multi-enzyme system was inferred from inhibition studies. The formation of catechol was inhibited by Mg2+, Zn2+, and Co2+ ions, whereas anthranilic acid disappearance was not affected to the same extent. The effect of metal chelating agents like EDTA, cyanide and pyrophosphate showed a similar trend. PCMB inhibited catechol formation but had no effect on anthranilic acid disappearance. The reaction was not inhibited by catalase, nor was it activated by peroxide-donating systems. This ruled out the possibility of peroxidative type of reaction. The overall reaction is markedly activated by NADPH and THFA. This multi-enzyme was separated into three different components, by fractionation with Alumina Cγ and calcium phosphate gels. The overall reaction catalysed by these components can be represented as anthranilic acid→3-hydroxy anthranilic acid→o-aminophenol→catechol.

Oxidation of catechol in plants : III. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The Km for catechol was determined as 4 × 10−4 Image . The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.

Purification of 3,5-Dichlorocatechol 1,2-Dioxygenase, a Nonheme Iron Dioxygenase and a Key Enzyme in the Biodegradation of a Herbicide, 2,4-Dichlorophenoxyacetic acid (2,4-D), from Pseudomonas cepacia CSV90

An enzyme which cleaves the benzene ring of 3,5-dichiorocatechol has been purified to homogeneity from Pseudomonas cepacia CSV90, grown with 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole carbon source. The enzyme was a nonheme ferric dioxygenase and catalyzed the intradiol cleavage of all the examined catechol derivatives, 3,5-dichlorocatechol having the highest specificity constant of 7.3 μM−1 s−1 in an air-saturated buffer. No extradiol-cleaving activity was observed. Thus, the enzyme was designated as 3,5-dichlorocatechol 1,2-dioxygenase. The molecular weight of the native enzyme was ascertained to be 56,000 by light scattering method, while the Mr value of the enzyme denatured with 6 M guanidine-HCl or sodium dodecyl sulfate was 29,000 or 31,600, respectively, suggesting that the enzyme was a homodimer. The iron content was estimated to be 0.89 mol per mole of enzyme. The enzyme was deep red and exhibited a broad absorption spectrum with a maximum at around 425 nm, which was bleached by sodium dithionite, and shifted to 515 nm upon anaerobic 3,5-dichlorocatechol binding. The catalytic constant and the Km values for 3,5-dichlorocatechol and oxygen were 34.7 s−1 and 4.4 and 652 μM, respectively, at pH 8 and 25°C. Some heavy metal ions, chelating agents and sulfhydryl reagents inhibited the activity. The NH2-terminal sequence was determined up to 44 amino acid residues and compared with those of the other catechol dioxygenases previously reported.

The design and synthesis of redox core-alpha amino acid composites based on thiol-disulfide exchange mechanism and a comparative study of their zinc abstraction potential from [CCXX] boxes in proteins

The design and synthesis of agents that can abstract zinc from their [CCXX] (C=cysteine; X=cysteine/histidine) boxes by thioldisulfide exchange-having as control, the redox parities of the core sulfur ligands of the reagent and the enzyme, has been illustrated, and their efficiency demonstrated by monitoring the inhibition of the transcription of calf thymus DNA by E. coli RNA polymerase, which harbors two zinc atoms in their [CCXX] boxes of which one is exchangeable. Maximum inhibition possible with removal of the exchangeable zinc was seen with redox-sulfanilamide-glutamate composite. In sharp contrast, normal chelating agents (EDTA, phenanthroline) even in a thousand fold excess showed only marginal inhibition, thus supporting an exchange mechanism for the metal removal. (C) 2002 Elsevier Science Ltd. All rights reserved.

Designing carboxymethyl cellulose based layer-by-layer capsules as a carrier for protein delivery

Stable hollow microcapsules composed of sodium carboxymethyl cellulose (CMC) and poly (allylamine hydrochloride) (PAH) were produced by layer-by-layer adsorption of polyelectrolytes onto CaCO 3 microparticles. Subsequently the core was removed by addition of chelating agents for calcium ions. Zeta potential studies showed charge reversal with deposition of successive polyelectrolyte layers, indicating that the alternate electrostatic adsorption of polyelectrolytes of opposite charge was successfully achieved. The size and surface morphology of the capsules was characterized by various microscopy techniques. The pH responsive loading behavior was elucidated by confocal laser scanning microscopy (CLSM) studies using fluorescence labeled dextran (FITC-dextran) and labeled BSA (FITC-BSA). CLSM images confirmed the open (pH ≤ 6) and closed state (pH ≥ 7) of the capsules. A model drug bovine serum albumin (BSA) was spontaneously loaded below its isoelectric point into hollow microcapsules, where BSA is positively charged. The loading of the BSA into the microcapsules was found to be dependent on the feeding concentration and pH of the medium. 65 of the loaded BSA was released over 7h of which about 34 was released in the first hour. These findings demonstrate that (CMC/PAH) 2 hollow capsules can be further exploited as a potential drug delivery system.

Polymorphic phase transition among the titania crystal structures using a solution-based approach: from precursor chemistry to nucleation process

Nanocrystalline titania are a robust candidate for various functional applications owing to its non-toxicity, cheap availability, ease of preparation and exceptional photochemical as well as thermal stability. The uniqueness in each lattice structure of titania leads to multifaceted physico-chemical and opto-electronic properties, which yield different functionalities and thus influence their performances in various green energy applications. The high temperature treatment for crystallizing titania triggers inevitable particle growth and the destruction of delicate nanostructural features. Thus, the preparation of crystalline titania with tunable phase/particle size/morphology at low to moderate temperatures using a solution-based approach has paved the way for further exciting areas of research. In this focused review, titania synthesis from hydrothermal/solvothermal method, conventional sol-gel method and sol-gel-assisted method via ultrasonication, photoillumination and ILs, thermolysis and microemulsion routes are discussed. These wet chemical methods have broader visibility, since multiple reaction parameters, such as precursor chemistry, surfactants, chelating agents, solvents, mineralizer, pH of the solution, aging time, reaction temperature/time, inorganic electrolytes, can be easily manipulated to tune the final physical structure. This review sheds light on the stabilization/phase transformation pathways of titania polymorphs like anatase, rutile, brookite and TiO2(B) under a variety of reaction conditions. The driving force for crystallization arising from complex species in solution coupled with pH of the solution and ion species facilitating the orientation of octahedral resulting in a crystalline phase are reviewed in detail. In addition to titanium halide/alkoxide, the nucleation of titania from other precursors like peroxo and layered titanates are also discussed. The nonaqueous route and ball milling-induced titania transformation is briefly outlined; moreover, the lacunae in understanding the concepts and future prospects in this exciting field are suggested.