Lanthanide chelate complexes of salicyloyl hydrazide-salicylaldehyde schiff base (SHSASB) and anthranilic acid-salicylaldehyde schiff base (AASASB)

New lanthanide complexes of salicylaldehyde-Schiff bases with salicyloyl hydrazide and anthranilic acid, were synthesized by a novel method consisting of refluxing the mixtures of Schiff base ligands and lanthanide trichloroacetate in acetone. Solid complexes of formulae Ln(SHSASB)s*2Hz0 and Ln2(AASASB)s*2Hz0 where Ln = La-Yb and Y, were isolated. Proton NMR and IR spectra for the complexes reveal the bidentate binding of both the Schiff base ligands to the lanthanide ion. Electronic spectra along with the conductance data for the complexes indicate a coordination number of six for the lanthanide ion in the complexes of both the Schiff bases.

Anthranilic acid hydroxylase from Aspergillus niger

The in vivo conversion of radioactive tryptophan to anthranilic acid and 2,3-dihydroxybenzoic acid by submerged cultures of Claviceps paspali was shown by Groeger and his co-workers (1965). More recently, Subba Rao et al. (1967a) reported that washed mycelial felts of Aspergillus niger incorporate the radioactivity from DL-tryptophan-C14 (benzene ring-labeled) into anthranilic acid, 3-hydroxyanthranilic acid, 2,3-dihydroxybenzoic acid and catechol. However, the conversion of anthranilic acid to 2,3-dihydroxybenzoic acid by cell-free preparations has not been demonstrated. In the present paper we report the demonstration of a soluble anthranilic acid hydroxylase from Aspergillus niger which is different from the anthranilic acid hydroxylases reported so far from microbes and higher plants.

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.

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.

Enzymatic conversion of anthranilic acid to catechol by chloroplast from the leaves of Tecoma stans

An enzyme system which converts anthranilic acid to catechol was detected in the leaves of Tecoma stans, and its properties studied. The system is present exclusively in the chloroplast fraction of the leaves. The optimum pH of the reaction is 5·2 and maximum activity was obtained with citrate-phosphate buffer. There was good stoichiometry between the amounts of anthranilic acid disappeared and the amounts of catechol and ammonia formed. The enzyme system showed an absolute requirement for oxygen and evidence was obtained for the probable participation of NADPH and FAD in the hydroxylation step. The optimum concentration of anthranilic acid was 10−4 M; at higher concentrations the reaction was inhibited to a considerable extent. Cyanide, pyrophosphate, and EDTA also caused inhibition indicating a requirement for metal ions.

Structural Studies of Analgesics and Their Interactions. VIII.* Rotational Isomerism and Disorder in the Crystal Structure of Meelofenamie Acid

Meclofenamic acid, C I4HIICI2NO2, probably the most potent among analgesic fenamates, crystallizes in the triclinic space group P1, with a = 8.569 (5), b = 8.954(8), c -- 9.371 (4) A, ct = 103.0 (2), fl -- 103.5 (2), y = 92.4 (2) ° , Z = 2, D m = 1.43 (4), D c = 1.41 Mg m -3. The structure was solved by direct methods and refined to R = 0.135 for 1062 observed reflections. The anthranilic acid moiety in the molecule is nearly planar and is nearly perpendicular to the 2,6-dichloro-3-methylphenyl group. The molecules, which exist as hydrogen-bonded dimers, have an internal hydrogen bond involving the imino and the carboxyl groups. The methyl group is disordered and occupies two positions with unequal occupancies. The disorder can be satisfactorily explained in terms of the rotational isomerism of the 2,6-dichloro-3-methylphenyl group about the bond which connects it to the anthranilic acid moiety and the observed occupancies on the basis of packing considerations.

Anthranilate Hydroxylase from Aspergillus niger: New Type of NADPH-Linked Nonheme Iron Monooxygenase

Anthranilate hydroxylase from Aspergillus niger catalyzes the oxidative deamination and dihydroxylation of anthranilic acid to 2,3-dihydroxybenzoic acid. This enzyme has been purified to homogeneity and has a molecular weight of 89,000. The enzyme is composed of two subunits of 42,000 with 2 gram-atoms of nonheme iron per mol. Fe2+-chelators like alpha,alpha'-dipyridyl and o-phenanthroline are potent inhibitors of the enzyme activity. Absorption and fluorescence spectra of the enzyme offer no evidence for the presence of other cofactors like flavin. Flavins and flavin-specific inhibitors like atebrin have no effect on the activity of the enzyme. The enzyme incorporates one atom of oxygen each from 18O2 and H218O into the product 2,3-dihydroxybenzoic acid. Based on these studies, it is concluded that anthranilate hydroxylase from A. niger is a new type of NADPH-linked nonheme iron monooxygenase.

Purification and Characterization of a New Indole Oxygenase from the Leaves of Tecoma stans L.

A new indole oxygenase from the leaves of Tecoma stans was isolated and purified to homogenity. The purified enzyme system catalyzes the conversion of indole to anthranilic acid. It is optimally active at pH 5.2 and 30°C. Two moles of oxygen are consumed and one mole of anthranilic acid is formed for every mole of indole oxidized. Dialysis resulted in complete loss of the activity. The inactive enzyme could be reactivated by the addition of concentrated dialysate. The enzyme is not inhibited by copperspecific chelators, non-heme iron chelators or atebrin. It is not a cuproflavoprotein, unlike the other indole oxygenases and oxidases.

Indolic compounds in the leaves of Tecoma stans

Indole, tryptophan, tryptamine and skatole were isolated from the leaves of Tecoma stans. Anthranilic acid was also identified in its free form, in contrast to its glucoside, in Jasminum grandiflorum. The presence of both indole and anthranilic acid in the leaves of Tecoma stans indicates that they are the true substrate and product of indole oxygenase from the leaves of Tecoma stans.

Indole oxygenase from the leaves of Tecoma stans

A new indole oxygenase from the leaves of Tecoma stans was isolated and purified to near homogeneity. The purified enzyme system catalyses the conversion of indole to anthranilic acid. It is optimally active at pH 5.2 and at 30°C. Oxygen (2 mol) is consumed and anthranilic acid (1 mol) is formed for every mole of indole oxidized. Neither sulfhydryl reagents nor sulfhydryl compounds inhibited the enzyme activity. The oxygenase also attacks, apart from indole, 5-hydroxyindole, 5-bromoindole and 5-methylindole. It is not inhibited by copper specific chelators or non-heme iron specific chelators. Atebrin did not inhibit the enzyme activity suggesting that it is not a flavoprotein, unlike other indole oxygenases and indole oxidases. Dialysis resulted in complete loss of enzyme activity. The inactive enzyme could not be reactivated by addition of various cofactors.

Degradation of substituted benzoic acids by a Micrococcus species

Abstract a Micrococcus sp. isolated by isophthalate enrichment, utilized 8 of the 13 substituted benzoic acids tested as the sole source of carbon and energy. The organism degraded benzoic acid and anthranilic acid through the intermediate formation of catechol. While salicylate is metabolized through genetisic acid, p-hydroxybenzoic acid is degraded through protocatechuic acid. The organism grew well on isophthalate but failed to utilize phthalate and terphthalate. Catechol disoxygenase, gentisate dioxygenase and protocatechuate dioxygenase activities were shown in the cell-free extracts. Catechol and protocatechuate are further metabolized through an ortho-cleavage pathway.

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.

Structural studies of analgesics and their interactions. XII. Structure and interactions of anti-inflammatory fenamates. A concerted crystallographic and theoretical conformational study

A theoretical conformational analysis of fenamates, which are N-arylated derivatives of anthranilic acid or 2-aminonicotinic acid with different substituents on the aryl (phenyl) group, is reported. The analysis of these analgesics, which are believed to act through the inhibition of prostaglandin biosynthesis, was carried out using semi-empirical potential functions. The results and available crystallographic observations have been critically examined in terms of their relevance to drug action. Crystallographic studies of these drugs and their complexes have revealed that the fenamate molecules share a striking invariant feature, namely, the sixmembered ring bearing the carboxyl group is coplanar with the carboxyl group and the bridging imino group,the coplanarity being stabilized by resonance interactions and an internal hydrogen bond between the imino and carboxyl groups. The results of the theoretical analysis provide a conformational rationale for the observed invariant coplanarity. The second sixmembered ring, which provides hydrophobicity in a substantial part of the molecule, has limited conformational flexibility in meclofenamic, mefenamic and flufenamic acids. Comparison of the conformational energy maps of these acids shows that they could all assume the same conformation when bound to the relevant enzyme. The present study provides a structural explanation for the difference in the activity of niflumic acid, which can assume a conformation in which the whole molecule is nearly planar. The main role of the carboxyl group appears to be to provide a site for intermolecular interactions in addition to helping in stabilizing the invariant coplanar feature and providing hydrophilicity at one end of the molecule. The fenamates thus provide a good example of conformation- dependent molecular asymmetry.

Indole oxygenase from the leaves of Jasminum grandiflorum

An indole oxygenase from the leaves of Jasminum grandiflorum was isolated and purified to near homogeneity. The purified enzyme system catalyses the conversion of indole to anthranilic acid. It is optimally active at pH 4.8 and at 30°C. Apart from indole, the oxygenase also attacks 5-hydroxy indole and 5-bromoindole. Both sulfhydryl reagents and sulfhydryl compounds inhibited the enzyme activity. Copper specific metal chelators such as salicylaldoxime, diethyl dithiocarbamate and neocuproine, inhibited the enzyme activity drastically. Inhibition caused by atebrine, could be reversed by FAD. Dialysis resulted in complete loss of enzyme activity. Inactive enzyme could be reactivated only by the addition of both FAD and Cu2+, suggesting that indole oxygenase is a cuproflavoprotein.

New pathway for the biodegradation of indole in Aspergillus niger

Indole and its derivatives form a class of toxic recalcitrant environmental pollutants. The growth of Aspergillus niger was inhibited by very low concentrations (0.005 to 0.02%) of indole, even when 125- to 500-fold excess glucose was present in the medium. When 0.02% indole was added, the fungus showed a lag phase for about 30 h and the uptake of glucose was inhibited. Indole was metabolized by a new pathway via indoxyl (3-hydroxyindole), N-formylanthranilic acid, anthranilic acid,2,3-dihydroxybenzoic acid, and catechol, which was further degraded by ortho cleavage. The enzymes N-formylanthranilate deformylase, anthranilate hydroxylase, 2,3-dihydroxybenzoate decarboxylase, and catechol dioxygenase were induced by indole as early as after 5 h of growth, and their activities were demonstrated in a cell-free system.