salicylates and 4-methylcatechol

The polyphenol oxidase (LsPPO) from a wild edible mushroom Lactarius salmonicolor was purified using a Sepharose 4B-L-tyrosine-p-amino benzoic acid affinity column. At the optimum pH and temperature, the K(M) and V(Max) values of LsPPO towards catechol, 4-methylcatechol and pyrogallol were determined as 0.025 M & 0.748 EU/mL, 1.809 x 10(- 3) M & 0.723 EU/mL and 9.465 x 10(- 3) M & 0.722 EU/mL, respectively. Optimum pH and temperature values of LsPPO for the three substrates above ranged between the pH 4.5-11.0 and 5-50 degrees C. Enzyme activity decreased due to heat denaturation with increasing temperature. Effects of a variety of classical PPO inhibitors were investigated opon the activity of LsPPO using catechol as the substrate. IC(50) values for glutathione, p-aminobenzenesulfonamide, L-cysteine, L-tyrosine, oxalic acid, beta-mercaptoethanol and syringic acid were determined as 9.1 x 10(- 4), 2.3 x 10(- 4) M, 1.5 x 10(- 4) M, 3.8 x 10(- 7) M, 1.2 x 10(- 4) M, 4.9 x 10(- 4) M, and 4 x 10(- 4) M respectively. Thus L-tyrosine was by far the most effective inhibitor. Interestingly, sulfosalicylic acid behaved as an activator of LsPPO in this study.

Topics: Agaricales; Benzenesulfonates; Catechol Oxidase; Catechols; Enzyme Inhibitors; Hydrogen-Ion Concentration; Kinetics; Pyrogallol; Salicylates; Substrate Specificity; Temperature; Tyrosine

Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.

Topics: Base Sequence; Catechol 1,2-Dioxygenase; Catechols; Intramolecular Lyases; Mixed Function Oxygenases; Molecular Sequence Data; Multigene Family; Pseudomonas; Reverse Transcriptase Polymerase Chain Reaction; Salicylates; Sorbic Acid

Pseudomonas putida CSV86, a soil bacterium, grows on 1- and 2-methylnaphthalene as the sole source of carbon and energy. In order to deduce the pathways for the biodegradation of 1- and 2-methylnaphthalene, metabolites were isolated from the spent medium and purified by thin layer chromatography. Emphasis has been placed on the structural characterisation of isolated intermediates by GC-MS, demonstration of enzyme activities in the cell free extracts and measurement of oxygen uptake by whole cells in the presence of various probable metabolic intermediates. The data obtained from such a study suggest the possibility of occurrence of multiple pathways in the degradation of 1- and 2-methylnaphthalene. We propose that, in one of the pathways, the aromatic ring adjacent to the one bearing the methyl moiety is oxidized leading to the formation of methylsalicylates and methylcatechols. In another pathway the methyl side chain is hydroxylated to -CH2-OH which is further converted to -CHO and -COOH resulting in the formation of naphthoic acid as the end product. In addition to this, 2-hydroxymethylnaphthalene formed by the hydroxylation of the methyl group of 2-methylnaphthalene undergoes aromatic ring hydroxylation. The resultant dihydrodiol is further oxidised by a series of enzyme catalysed reactions to form 4-hydroxymethyl catechol as the end product of the pathway.

Topics: Biodegradation, Environmental; Carboxylic Acids; Catechols; Hydroxylation; Naphthalenes; Pseudomonas putida; Salicylates