salicylates and anthranilic-acid

Indole, an important signaling molecule as well as a typical N-heterocyclic aromatic pollutant, is widespread in nature. However, the biotransformation mechanisms of indole are still poorly studied. Here, we sought to unlock the genetic determinants of indole biotransformation in strain Cupriavidus sp. SHE based on genomics, proteomics and functional studies. A total of 177 proteins were notably altered (118 up- and 59 downregulated) in cells grown in indole mineral salt medium when compared with that in sodium citrate medium. RT-qPCR and gene knockout assays demonstrated that an indole oxygenase gene cluster was responsible for the indole upstream metabolism. A functional indole oxygenase, termed IndA, was identified in the cluster, and its catalytic efficiency was higher than those of previously reported indole oxidation enzymes. Furthermore, the indole downstream metabolism was found to proceed via the atypical CoA-thioester pathway rather than conventional gentisate and salicylate pathways. This unusual pathway was catalyzed by a conserved 2-aminobenzoyl-CoA gene cluster, among which the 2-aminobenzoyl-CoA ligase initiated anthranilate transformation. This study unveils the genetic determinants of indole biotransformation and will provide new insights into our understanding of indole biodegradation in natural environments and its functional studies.

Topics: Acyl Coenzyme A; Biodegradation, Environmental; Biotransformation; Cupriavidus; Dioxygenases; Down-Regulation; Gene Deletion; Genomics; Gentisates; Indoles; Multigene Family; ortho-Aminobenzoates; Proteomics; Salicylates; Up-Regulation

Anthranilate phosphoribosyltransferase (AnPRT, EC 2.4.2.18) is a homodimeric enzyme that catalyzes the reaction between 5'-phosphoribosyl 1'-pyrophosphate (PRPP) and anthranilate, as part of the tryptophan biosynthesis pathway. Here we present the results of the first chemical screen for inhibitors against Mycobacterium tuberculosis AnPRT (Mtb-AnPRT), along with crystal structures of Mtb-AnPRT in complex with PRPP and several inhibitors. Previous work revealed that PRPP is bound at the base of a deep cleft in Mtb-AnPRT and predicted two anthranilate binding sites along the tunnel leading to the PRPP binding site. Unexpectedly, the inhibitors presented here almost exclusively bound at the entrance of the tunnel, in the presumed noncatalytic anthranilate binding site, previously hypothesized to have a role in substrate capture. The potencies of the inhibitors were measured, yielding Ki values of 1.5-119 μM, with the strongest inhibition displayed by a bianthranilate compound that makes hydrogen bond and salt bridge contacts with Mtb-AnPRT via its carboxyl groups. Our results reveal how the substrate capture mechanism of AnPRT can be exploited to inhibit the enzyme's activity and provide a scaffold for the design of improved Mtb-AnPRT inhibitors that may ultimately form the basis of new antituberculosis drugs with a novel mode of action.

Topics: Anthranilate Phosphoribosyltransferase; Antitubercular Agents; Binding Sites; Catalytic Domain; Crystallography, X-Ray; Drug Evaluation, Preclinical; Enzyme Inhibitors; Kinetics; Models, Molecular; Mycobacterium tuberculosis; ortho-Aminobenzoates; Phosphoribosyl Pyrophosphate; Substrate Specificity

Mechanisms by which dissolved organic matter (DOM) mediates the photochemical reduction of Hg(II) in aquatic ecosystems are not fully understood, owing to the heterogeneous nature and complex structural properties of DOM. In this work, naturally occurring aromatic compounds including salicylic, 4-hydrobenzoic, anthranilic, 4-aminobenzoic, and phthalic acid were systematically studied as surrogates for DOM in order to gain an improved mechanistic understanding of these compounds in the photoreduction of Hg(II) in water. We show that the photoreduction rates of Hg(II) are influenced not only by the substituent functional groups such as -OH, -NH(2) and -COOH on the benzene ring, but also the positioning of these functional groups on the ring structure. The Hg(II) photoreduction rate decreases in the order anthranilic acid>salicylic acid>phthalic acid according to the presence of the -NH(2), -OH, -COOH functional groups on benzoic acid. The substitution position of the functional groups affects reduction rates in the order anthranilic acid>4-aminobenzoic acid and salicylic acid>4-hydroxybenzoic acid. Reduction rates correlate strongly with ultraviolet (UV) absorption of these compounds and their concentrations, suggesting that the formation of organic free radicals during photolysis of these compounds is responsible for Hg(II) photoreduction. These results provide insight into the role of low-molecular-weight organic compounds and possibly DOM in Hg photoredox transformation and may thus have important implications for understanding Hg geochemical cycling in the environment.

Topics: 4-Aminobenzoic Acid; Mercury Compounds; Molecular Weight; ortho-Aminobenzoates; Oxidation-Reduction; Parabens; Photolysis; Phthalic Acids; Salicylates; Water; Water Pollutants, Chemical

Acridone alkaloids formed by acridone synthase in Ruta graveolens L. are composed of N-methylanthraniloyl CoA and malonyl CoAs. A 1095 bp cDNA from elicited Ruta cells was expressed in Escherichia coli, and shown to encode S-adenosyl-l-methionine-dependent anthranilate N-methyltransferase. SDS-PAGE of the purified enzyme revealed a mass of 40 +/- 2 kDa, corresponding to 40 059 Da for the translated polypeptide, whereas the catalytic activity was assigned to a homodimer. Alignments revealed closest relationships to catechol or caffeate O-methyltransferases at 56% and 55% identity (73% similarity), respectively, with little similarity ( approximately 20%) to N-methyltransferases for purines, putrescine, glycine, or nicotinic acid substrates. Notably, a single Asn residue replacing Glu that is conserved in caffeate O-methyltransferases determines the catalytic efficiency. The recombinant enzyme showed narrow specificity for anthranilate, and did not methylate catechol, salicylate, caffeate, or 3- and 4-aminobenzoate. Moreover, anthraniloyl CoA was not accepted. As Ruta graveolens acridone synthase also does not accept anthraniloyl CoA as a starter substrate, the anthranilate N-methylation prior to CoA activation is a key step in acridone alkaloid formation, channelling anthranilate from primary into secondary branch pathways, and holds promise for biotechnological applications. RT-PCR amplifications and Western blotting revealed expression of the N-methyltransferase in all organs of Ruta plants, particularly in the flower and root, mainly associated with vascular tissues. This expression correlated with the pattern reported previously for expression of acridone synthase and acridone alkaloid accumulation.

Topics: 4-Aminobenzoic Acid; Acridines; Acridones; Amino Acid Sequence; Blotting, Western; Catechols; Methyltransferases; Molecular Sequence Data; Molecular Structure; ortho-Aminobenzoates; Plant Proteins; Polymerase Chain Reaction; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Rutaceae; Salicylates; Substrate Specificity

The unicellular flagellate Euglena gracilis was used in order to assess the inhibition of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and N-methyl-N-nitrosourea (MNU) mutagenicities, which induce white mutants due to the irreversible loss of chloroplasts. All tested compounds, including o-aminobenzoic acid and p-aminobenzoic acid, salicylic acid, acetylsalicylic acid, sodium salicylate and p-aminosalicylic acid, were not mutagenic per se and inhibited MNNG mutagenicity by at least 50%. The last two compounds inhibited by at least 50% also MNU mutagenicity.

Topics: 4-Aminobenzoic Acid; Aminosalicylic Acid; Animals; Antimutagenic Agents; Aspirin; Chloroplasts; Euglena gracilis; Eukaryotic Cells; Methylnitronitrosoguanidine; Methylnitrosourea; Mutagenicity Tests; Mutagens; Mutation; ortho-Aminobenzoates; Pigmentation; Salicylates; Salicylic Acid; Sodium Salicylate

The pH dependence of the redox behavior of anthranilate hydroxylase from Trichosporon cutaneum in its uncomplexed and anthranilate-complexed forms, as well as the effects on the reduction potential, at pH 7.4, of enzyme in complex with 3-methylanthranilate, salicylate, 3-acetylpyridine adenine dinucleotide phosphates, and azide plus anthranilate, is described. At pH 7.4 the midpoint potential of uncomplexed enzyme (EFlox/EFlredH-) is -0.229 V vs SHE, close to that of free flavin. The aromatic substrates and effector all shift the midpoint potential value in a positive direction by 0.068-0.100 V. This shift results in thermodynamically more favorable reduction of the substrate/effector-complexed enzyme by NADPH. Consistent with thermodynamic considerations, the aromatic substrates (or effector) are bound to the reduced enzyme 2-4 orders of magnitude more tightly than to the oxidized enzyme. The tighter binding of the substrate to the two-electron-reduced enzyme may be related to the double hydroxylation reaction performed by this enzyme, which is a more complex reaction than is carried out by typical flavoprotein hydroxylases. The acetylpyridine nucleotides appear to have no significant regulatory role.

Topics: Azides; Binding Sites; Hydrogen-Ion Concentration; Kinetics; Mitosporic Fungi; Mixed Function Oxygenases; NADP; ortho-Aminobenzoates; Oxidation-Reduction; Salicylates; Salicylic Acid; Thermodynamics; Trichosporon

Rapid reaction kinetics of the flavoprotein anthranilate hydroxylase from Trichosporon cutaneum were examined for reactions involving anthranilate, the native substrate. As was reported earlier for the nonhydroxylated substrate analogue, salicylate, some reactions in the first turnover with anthranilate occur slower than those in subsequent turnovers (Powlowski, J., Massey, V., and Ballou, D. P. (1989) J. Biol. Chem. 264, 5606-5612). Evidence is presented for slow conformational changes that occur both on binding of the aromatic ligand and on reduction of the enzyme. These changes are apparently important for rapid anthranilate binding to occur in turnovers subsequent to the first. Moreover, bound anthranilate is required for rapid reduction of enzyme-bound FAD by NADPH. Studies to probe the accessibility of reagents to modified flavins that had been incorporated into the apoenzyme indicate that anthranilate binding causes a conformational change in the protein, allowing increased access to the benzene ring moiety of the flavin. An unusual isotope effect with (R)-NADPD (4(R)-2H] NADPH) is observed on Kd rather than on kred, which is consistent with a model involving slow interconversion of enzyme-substrate complexes before productive binding of NADPH and reduction of the enzyme flavin.

Topics: Binding, Competitive; Kinetics; Ligands; Mathematics; Mitosporic Fungi; Mixed Function Oxygenases; Models, Theoretical; ortho-Aminobenzoates; Oxidation-Reduction; Protein Conformation; Salicylates; Salicylic Acid; Spectrophotometry; Trichosporon

Topics: Anilides; Animals; Anti-Inflammatory Agents; Antimetabolites; Azides; Benzoates; Cartilage; Cattle; Connective Tissue; Hydroxamic Acids; Liver; Manometry; Metabolism; Mitochondria; Mitochondria, Liver; Naphthalenes; Nitrophenols; ortho-Aminobenzoates; Oxidative Phosphorylation; Pharmacology; Phosphates; Phosphorus Isotopes; Piperidines; Rats; Research; Salicylamides; Salicylates; Sulfates; Sulfur Isotopes; Trachea

Topics: Antioxidants; Antirheumatic Agents; Glycyrrhiza; Humans; Metabolism; ortho-Aminobenzoates; Oxidative Phosphorylation; Phenylbutazone; Quinolines; Salicylates

The effects of eighteen substituted benzoic acids on the rate of oxygen consumption have been studied in rats. 2:3-Dihydroxybenzoic acid, phthalic acid and 6-methylsalicylic acid were, at the doses used, inactive; m- and p-hydroxybenzoic acid, 2:4-, 2:5-, 2:6-, 3:4-, and 3:5-dihydroxybenzoic acid, o-aminobenzoic acid, salicyluric acid, salicylamide and 5-aminosalicylic acid decreased the rate of oxygen consumption. Only salicylic acid and o-, m- and p-cresotic acid (3-, 4- and 5-methylsalicylic acid respectively) increased the rate of oxygen consumption. Molar potency ratios of the cresotic acids as metabolic stimulants relative to salicylic acid were determined; o-cresotic acid was the most powerful with a ratio of 2.61, m- and pcresotic acid had values of 1.78 and 1.89 respectively. Two possible explanations of the higher potencies of the cresotic acids were considered. No difference in the primary action of the drug was established by determining the effect on rate of oxygen consumption of a mixture of o-cresotic and salicylic acids. An alternative possibility was that the rates of detoxication and excretion of the cresotic acids differed among themselves and from salicylic acid. No such differences were found.

Topics: Animals; Benzoates; Central Nervous System Agents; Central Nervous System Stimulants; Hippurates; Hydroxybenzoates; Male; Mesalamine; Metabolism; ortho-Aminobenzoates; Rats; Salicylates