ABSTRACT
The growth of the white-rot basidiomycete Pleurotus sajor-caju in malt-agar plates was inhibited by three naturally occurring, plant-derived naphthoquinones: juglone, lawsone, and plumbagin. The latter two compounds exerted the most potent antifungal activity, and lawsone killed the mycelium at concentrations higher than 200 ppm. Plates containing juglone and lawsone presented large decolorized areas extending from area of fungal growth, suggesting an extracellular enzymatic degradation of these quinones. Screening of culture plates for extracellular enzymatic activities revealed the presence of both laccase and veratryl alcohol oxidase in most plates, the diffusion of both enzymes matching the decolorized area. In agitated cultures, the presence of juglone was found to stimulate the production of veratryl alcohol oxidase in a significant manner. This is the first time degradation of plant derived naphthoquinones by a white-rot fungus is reported.
Subject(s)
Naphthoquinones/metabolism , Naphthoquinones/pharmacology , Plants/chemistry , Pleurotus/drug effects , Pleurotus/growth & development , Biodegradation, Environmental , Culture Media , Microbial Sensitivity TestsABSTRACT
Tyrosinase is a copper containing protein which catalyzes the hydroxylation of monophenols and the oxidation of diphenols to o-quinones. The monophenolase activity of tyrosinase is characterized by a typical lag time. In this paper the influence of 3-hydroxyanthranilic acid on monophenolase activity of tyrosinase is reported. 3-Hydroxyanthranilic acid reduced the lag time of tyrosinase when the enzyme acted on N-acetyl-L-tyrosine and on 4-tert-butylphenol. In the presence of 3-hydroxyanthranilic acid, the reaction product 4-tert-butyl-o-benzoquinone, derived from 4-tert-butylphenol oxidation, was formed at a higher rate than in its absence. The results reported in this paper indicate that 3-hydroxyanthranilic acid could affect the enzymic activity of mushroom tyrosinase probably by acting as a diphenol substrate. A K(m) value of 0.78 mM was calculated for 3-hydroxyanthranilic acid as substrate. When tyrosinase acted on 4-tert-butylphenol, K(m) for 3-hydroxyanthranilic acid as a cofactor was estimated to be 37.5 microM. No effect was observed on the diphenolase activity of the enzyme acting on 4-tert-butylcatechol in the presence of 3-hydroxyanthranilic acid.
Subject(s)
3-Hydroxyanthranilic Acid/pharmacology , Basidiomycota/enzymology , Free Radical Scavengers/pharmacology , Fungal Proteins/metabolism , Monophenol Monooxygenase/metabolism , Chromatography, High Pressure Liquid , Fungal Proteins/drug effects , Monophenol Monooxygenase/drug effects , Monophenol Monooxygenase/isolation & purification , Tyrosine/analogs & derivatives , Tyrosine/metabolismABSTRACT
Copper amine oxidases utilize 2,4,5-trihydroxyphenylalanine quinone (topaquinone) as a cofactor in enzymatic catalysis. This cofactor is formed from a tyrosine residue through a self-catalytic mechanism with the participation of the copper ion at the active site. Although pathways have been postulated for topaquinone biogenesis, portions of this scheme are still unclear. We utilized 4-tert-butyl-derived models for the putative intermediates of topaquinone generation and studied the effect of Cu(II) and Zn(II) ions on each autoxidative step from dopa- to topaquinone-like compounds at physiological pH (7.4). Several polyvinyl-alcohol-based soluble resins bearing mono- and di-hydroxyphenolic moieties were also prepared, and their tendency to give hydroxyquinonic structures when incubated at alkaline pH values was investigated. Our results confirm (although indirectly) the formation of dopa and dopaquinone during topaquinone biosynthesis. Moreover, we collected evidence that, following the formation of dopa, the role of the active-site copper ion in topaquinone biogenesis would be limited to the catalysis of the two subsequent quinonization steps (i.e. from dopa to dopaquinone and from topa to topaquinone), thus disfavoring the possibility of a direct intervention of the metal ion in the hydroxylation of dopaquinone. In particular, Cu(II) was shown to influence deeply the autoxidation of 1,2,5-trihydroxy-4-tert-butylbenzene, used as model of topa, both increasing the reaction rate and changing its mechanism. The mechanistic implications of these findings for the biogenesis of topaquinone and its analogs at the active site of various amine oxidases are discussed.
Subject(s)
Amine Oxidase (Copper-Containing)/chemistry , Amine Oxidase (Copper-Containing)/metabolism , Dihydroxyphenylalanine/analogs & derivatives , Aldehydes/chemistry , Aldehydes/metabolism , Benzene Derivatives/chemistry , Benzene Derivatives/metabolism , Benzoquinones/chemistry , Benzoquinones/metabolism , Binding Sites , Catalase/chemistry , Catalase/metabolism , Catalase/pharmacology , Catechols/chemistry , Catechols/metabolism , Copper/chemistry , Copper/metabolism , Copper/pharmacology , Dihydroxyphenylalanine/biosynthesis , Dihydroxyphenylalanine/metabolism , Free Radical Scavengers/chemistry , Free Radical Scavengers/metabolism , Free Radical Scavengers/pharmacology , Hydroxylation , Oxidation-Reduction/drug effects , Structure-Activity Relationship , Superoxide Dismutase/chemistry , Superoxide Dismutase/metabolism , Superoxide Dismutase/pharmacology , Zinc/chemistry , Zinc/metabolism , Zinc/pharmacologyABSTRACT
An analytical method allowing the detection of polyphenol oxidase activity on polyacrylamide gel electrophoresis (PAGE) is described. The method is rapid, sensitive and specific and is based on a coupling reaction between 4-tert-butyl-o-benzoquinone and the aromatic amine, 4-amino-N,N-diethylaniline sulphate. Catecholase activity of polyphenol oxidase appears as blue stained bands on a colourless background.
Subject(s)
Catechol Oxidase/analysis , Electrophoresis, Polyacrylamide Gel/methods , Animals , Humans , Sensitivity and Specificity , Staining and LabelingABSTRACT
A simple model, 4-tert-butyl-1,2-benzoquinone, was chosen to study the hydroxylation step of the tyrosine-derived Dopaquinone residue at the active site of copper amine oxidases in the self-catalytic generation of the Topaquinone cofactor. This hydroxylation step was studied both in the presence and absence of free copper(II), and was found to be dependent on pH value but not on the presence of metal ions. It is therefore proposed that, hydroxide ion and not water should be the true reactive species in this key biosynthetic step of the Topaquinone cofactor, and that the active site Cu2+ is implied, at this point of cofactor biosynthesis, in the quinonisation of Topa rather than in the hydroxylation of Dopaquinone.
Subject(s)
Amine Oxidase (Copper-Containing)/metabolism , Benzoquinones/metabolism , Dihydroxyphenylalanine/analogs & derivatives , Dihydroxyphenylalanine/metabolism , Hydroxylation , Models, ChemicalABSTRACT
An NAD(P)H:(quinone acceptor) oxidoreductase (EC 1.6.99.2) was purified from Glycine max seedlings by means of chromatographic procedures. After 1371-fold purification, the enzyme showed a single band in IEF corresponding to an isoelectric point of 6.1. A single band was also found in native-PAGE both by activity staining and Coomassie brilliant blue staining. The molecular mass determined in SDS-PAGE was 21900 Da, while in HPLC gel-filtration it was 61000 Da. The NAD(P)H:quinone oxidoreductase was able to use NADH or NADPH as the electron donor. Among the artificial quinones which are reduced by this enzyme, 6-hydroxydopa- and 6-hydroxydopamine-quinone are of particular interest because of their neurotoxic effects.
