Inhibition of mushroom tyrosinase by tropolone

Tropolone inhibits both mono- and o-dihydroxyphenolase activity of mushroom tyrosinase. Most of the inhibition exerted by tropolone was reversed by dialysis or by excess CU²⁺. The data indicate that tropolone and o-dihydroxyphenols compete for binding to the copper at the active site of the enzyme. Comparison between the effectiveness of various copper chelators showed that tropolone is one of the most potent inhibitors of mushroom tyrosinase; 50% inhibition was observed with 0.4 × 10^?6 M tropolone.     

Role of tyrosinase in the genoprotective effect of the edible mushroom, Agaricus bisporus

A heat-labile protein has been identified in fruit bodies of the edible mushroom, Agaricus bisporus, which protects Raji cells (a human lymphoma cell line) against H2O2-induced oxidative damage to cellular DNA. This protein has been purified following salt fractionation, combined with ion-exchange, hydrophobic interaction and adsorption chromatography. Based on catalytic and electrophoretic properties, and inhibition studies using tropolone, the protein was identified as tyrosinase. The genoprotective effect of A. bisporus tyrosinase, determined using the single-cell gel electrophoresis met") assay, has been shown to be dependent upon the enzymic hydroxylation of tyrosine to L-DOPA and subsequent conversion of this metabolite to dopaquinone. The possible role of dopaquinone, and other L-DOPA oxidation products, in enhancing the cellular antioxidant defence mechanisms is discussed.     

Inhibitory effects of phloridzin dihydrate on the activity of mushroom (Agaricus bisporus) tyrosinase

The inhibitory effects of phloridzin dihydrate on the activity of mushroom tyrosinase have been studied. The results show that phloridzin can inhibit the diphenolase activity of the enzyme and the inhibition displays to be reversible. The IC(50) value was estimated as 110microM. The kinetic analysis showed that the inhibition of phloridzin on the diphenolase activity of the enzyme is of competitive type, and the inhibition constant (K(I)) was determined to be 64.3microM. The inhibitory effects of the different concentrations of phloridzin on the monophenolase activity were also studied. There were almost no changes in the lag period and the steady-state rate, while the plateaus in the inhibitory curve lowered with increasing the concentration of phloridzin when using tyrosine as a substrate.     

Irreversibly inhibitory kinetics of 3,5-dihydroxyphenyl decanoate on mushroom (Agaricus bisporus) tyrosinase

3,5-Dihydroxyphenyl decanoate (DPD) is found to inhibit the diphenolase activity of tyrosinase from mushroom (Agaricus bisporus). The effects of DPD on the diphenolase activity of mushroom tyrosinase have been studied. The results show that the enzyme activity decreases very slowly with an increase in DPD concentrations at lower concentrations of DPD (between 5 and 60 microM). But at higher concentrations of DPD, DPD can strongly inhibit the diphenolase activity of the enzyme and the inhibition is irreversible. The IC50 value was estimated to be 96.5 microM. The inhibition mechanism of DPD has been investigated and the results show that DPD can bind to the free enzyme molecule and enzyme-substrate complex and lose the enzyme activity completely. The inhibition kinetics has been studied in detail by using the kinetic method of the substrate reaction described by Tsou. The microscopic rate constants of the enzyme inhibited by DPD at higher concentrations have been determined.     

RAPD discrimination of Agaricus bisporus mushroom cultivars

Cultivars of the white button mushroom Agaricus bisporus are difficult to differentiate, which has made strain protection problematic for this crop species. We have used RAPDs to discriminate between 26 strains of A. bisporus, 24 of which were commercial cultivars, and to characterise the genetic relatedness of these strains. Using 20 primers, 211 RAPD markers were identified and used in hierarchical cluster, patristic distance and parsimony analyses. All strains could be differentiated using the aggregated primer data. Although no one primer could differentiate all 26 strains, several individual primers yielded unique fingerprints for a variety of strains. The greatest differences (up to 28% variation) were observed in comparisons with or between two wild collections of A. bisporus. Quondam cultivars, commercial brown and off-white varieties proved more variable than the widely grown 'hybrid' types. Of the 15 hybrid varieties analysed, only one differed substantially (20% or more variable). The patristic and parsimony analyses both demonstrated the gross similarity of the hybrids, many of which appear to be essentially derived varieties from two original hybrid cultivars. RAPD analyses can assist mushroom strain identification and could play a role in the protection of novel cultivars.     

Inhibitory effect of 4-cyanobenzaldehyde and 4-cyanobenzoic acid on mushroom (Agaricus bisporus) tyrosinase

Mushroom tyrosinase (EC 1.14.18.1), a copper containing oxidase, catalyzes both the hydroxylation of tyrosine into o-diphenols and the oxidation of o-diphenols into o-quinones. In the current study, the effects of 4-cyanobenzaldehyde and 4-cyanobenzoic acid on the monophenolase and diphenolase activities of mushroom tyrosinase have been studied. The results show that 4-cyanobenzaldehyde and 4-cyanobenzoic acid can inhibit both the monophenolase activity and the diphenolase activity of mushroom tyrosinase. The lag phase of tyrosine oxidation catalyzed by the enzyme was obviously lengthened, and the steady-state activity of the enzyme decreased sharply. 1.0 mM 4-cyanobenzaldehyde and 4-cyanobenzoic acid can lengthen the lag phase from 78 s to 134 and 115 s, respectively. Both 4-cyanobenzaldehyde and 4-cyanobenzoic acid can lead to reversible inhibition of the enzyme. The IC50 values of 4-cyanobenzaldehyde and 4-cyanobenzoic acid were estimated as 0.62 and 2.45 mM for monophenolase and as 0.72 and 1.40 mM for diphenolase, respectively. A kinetic analysis shows that 4-cyanobenzaldehyde and 4-cyanobenzoic acid are mixed-type inhibitors for the diphenolase. The apparent inhibition constants for 4-cyanobenzaldehyde and 4-cyanobenzoic acid binding with both the free enzyme and the enzyme-substrate complex have been determined and compared.     

Purine degradation in the edible mushroom Agaricus bisporus

Agaricus bisporus is able to use urate, allantoin, allantoate, urea and alloxanate as nitrogen sources for growth. The presence of urate oxidase, allantoinase, ureidoglycolase and urease activities, both in fruit bodies and mycelia, points to a degradative pathway for urate similar to that found in various microorganisms. So far all efforts, to demonstrate the enzyme responsible for allantoate degradation failed. A urease inhibitor appeared to be present in cell-free extracts, from fruit bodies.     

Microbial ecology of the Agaricus bisporus mushroom cropping process

Applied Microbiology and Biotechnology - Agaricus bisporus is the most widely cultivated mushroom species in the world. Cultivation is commenced by inoculating beds of semi-pasteurised composted...     

The metabolic pathway catalyzed by the tyrosinase of Agaricus bisporus

N-t-Butyloxycarbonyl-gamma-L-glutaminyl-2-bromo-4-hydroxybenzene alpha-benzyl ester was synthesized as a precursor to gamma-L-glutaminyl-4-hydroxy[2-3H]benzene. With this labeled compound and the previously synthesized gamma-L-glutaminyl-4-hydroxy[3,5-3H]benzene, the stoichiometry of ring substitution was determined for the tyrosinase-catalyzed metabolic pathway of Agaricus bisporus. In this pathway, gamma-L-glutaminyl-4-hydroxybenzene is hydroxylated to gamma-L-glutaminyl-3,4-dihydroxybenzene which is oxidized to gamma-L-glutaminyl-3,4-benzoquinone and a compound of previously unknown structure, "490." The results indicated that the "490" quinone was derived from gamma-L-glutaminyl-3,4-benzoquinone without further ring substitution. A base-catalyzed, nonenzymatic reaction of gamma-L-glutaminyl-3,4-benzoquinone was observed which yielded a compound with a 490 nm chromophore. gamma-Glutamyl transpeptidase cleavage of gamma-L-glutaminyl-3,4-dihydroxybenzene led to the release of 4-aminocatechol which air-oxidized to a compound with identical spectral properties to "490." The structure of "490" was thus determined to be 2-hydroxy-4-imino-2,5-cyclohexadiene-1-one(2-hydroxy-4-iminoquinone). The tyrosinase-catalyzed hydroxylation of gamma-L-glutaminyl-4-hydroxybenzene was found to be optimal at pH 8.0, while the enzymatic oxidation of gamma-L-glutaminyl-3,4-dihydroxybenzene was optimal at pH 6.0.     

Volatile compounds from the mycelium of the mushroom Agaricus bisporus

The pattern of volatiles from the mycelium of two commercial strains of Agaricus bisporus, grown in axenic culture on a semi-synthetic medium, was found to be broadly similar to that of the volatiles identified from sporophores. Tetrachloro-1,4-dimethoxybenzene, a known secondary metabolite of several Basidiomycetes, was found in the mycelium though not in the sporophores. [³⁶Cl]Tetrachloro-1,4-dimethoxybenzene was obtained when sodium [¹³Cl]chloride was added to the medium.     

DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus.

Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spore descent. Homokaryons recovered from protoplast regenerants of heterokaryotic strains carried a subset of the RAPD markers found in the heterokaryon, and both of the haploid nuclei from two heterokaryons were distinguishable. RAPD markers also served to verify the creation of a hybrid heterokaryon and to analyze meiotic progeny from this new strain: most of the basidiospores displayed RAPD fingerprints identical to that of the parental heterokaryon, although a few selected slow growers were homoallelic at a number of loci that were heteroallelic in the parent, suggesting that they represented rare homokaryotic basidiospores; crossover events between a RAPD marker locus and its respective centromere appeared to be infrequent. These results demonstrate that RAPD markers provide an efficient alternative for strain fingerprinting and a versatile tool for genetic studies and manipulations of A. bisporus.     

DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus.

Single 10-bp primers were used to generate random amplified polymorphic DNA (RAPD) markers from commercial and wild strains of the cultivated mushroom Agaricus bisporus via the polymerase chain reaction. Of 20 primers tested, 19 amplified A. bisporus DNA, each producing 5 to 15 scorable markers ranging from 0.5 to 3.0 kbp. RAPD markers identified seven distinct genotypes among eight heterokaryotic strains; two of the commercial strains were shown to be related to each other through single-spore descent. Homokaryons recovered from protoplast regenerants of heterokaryotic strains carried a subset of the RAPD markers found in the heterokaryon, and both of the haploid nuclei from two heterokaryons were distinguishable. RAPD markers also served to verify the creation of a hybrid heterokaryon and to analyze meiotic progeny from this new strain: most of the basidiospores displayed RAPD fingerprints identical to that of the parental heterokaryon, although a few selected slow growers were homoallelic at a number of loci that were heteroallelic in the parent, suggesting that they represented rare homokaryotic basidiospores; crossover events between a RAPD marker locus and its respective centromere appeared to be infrequent. These results demonstrate that RAPD markers provide an efficient alternative for strain fingerprinting and a versatile tool for genetic studies and manipulations of A. bisporus.     

Quaternary structure of mushroom tyrosinase

The quaternary structure of $\text{Agaricus}$$\text{bispora}$ tyrosinase has been investigated by sodium dodecylsulfate-acrylamide gel electrophoresis. The enzyme was found to contain two types of polypeptide chains, referred to as Heavy, molecular weight 43,000 ± 1,000, and Light, molecular weight 13,400 ± 600. In aqueous solution the predominant form of tyrosinase m.w. 120,000, has the quaternary structure L₂H₂.     

Mitochondrial DNA variation in natural populations of the mushroom Agaricus bisporus

We investigated the patterns of mitochondrial DNA variation in the global population of the commercial mushroom Agaricus bisporus . Through the analysis of RFLP's among 441 isolates from nine countries in North America and Eurasia, we found a total of 140 mtDNA haplotypes. Based on population genetic analysis, there are four genetically distinct natural populations in this species, found in coastal California, desert California, France and Alberta (Canada). While 134 of the 140 mtDNA haplotypes were unique to single geographical regions, two mtDNA haplotypes, mt001 and mt002, were found in almost every population surveyed. These two mtDNA haplotypes also predominate among cultivars used throughout the world for at least the last two decades. These two mtDNA haplotypes are more similar to the cosmopolitan groups of mtDNA haplotypes than to the indigeneous clusters of mtDNA haplotypes from the two Californian regions.     

Purification and characterization of glutamine synthetase from the commerical mushroom Agaricus bisporus

Agaricus bisporus glutamine synthetase, a key enzyme in nitrogen metabolism, was purified to apparent homogeneity. The native enzyme appeared to be a GS-II type enzyme. It has a molecular weight of 325 kDa and consists of eight 46-kDa subunits. Its pI was found at 4.9. Optimal activity was found at 30°C. The enzyme had low thermostability. Stability declined rapidly at temperatures above 20°C. The enzyme exhibits a K _m for glutamate, ammonium, and ATP of 22mm, 0.16mm and 1.25mm respectively in the biosynthetic reaction, with optimal activity at pH 7. The enzyme is slightly inhibited by 10mm concentrations of l-alanine, l-histidine, l-tryptophan, anthranilic acid, and 5-AMP and was strongly inhibited by methionine sulfoximine and phosphinothricine. For the transferase reaction K _i-values were 890 m and 240 m for methionine sulfoximine and phosphinothricine respectively. For the biosynthetic reaction K _i was 17 m for both methionine sulfoximine and phosphinothricine.     

A life cycle assessment of Agaricus bisporus mushroom production in the USA

The International Journal of Life Cycle Assessment - Stakeholders across the food product supply chain are increasingly interested in understanding the environmental effects of food production....     

Purification and characterization of an acid phosphatase from the commercial mushroom Agaricus bisporus

Acid phosphatase [AP; EC 3.1.3.2], a key enzyme involved in the synthesis of mannitol in Agaricus bisporus, was purified to homogeneity and characterized. The native enzyme appeared to be a high molecular weight type glycoprotein. It has a molecular weight of 145 kDa and consists of four identical 39-kDa subunits. The isoelectric point of the enzyme was found at 4.7. Maximum activity occurred at 65°C. The optimum pH range was between 3.5 and 5.5, with maximum activity at pH 4.75. The enzyme was unaffected by EDTA, and inhibited by tartrate and inorganic phosphate. The enzyme exhibits a K _m for p-nitrophenylphosphate and fructose-6-phosphate of 370 M and 3.1 mM, respectively. A broad substrate specificity was observed with significant activities for fructose-6-phosphate, glucose-6-phosphate, mannitol-1-phosphate, AMP and -glycerol phosphate. Only phosphomonoesters were dephosphorylated. Antibodies raised against the purified enzyme could precipitate AP activity from a cell-free extract in an anticatalytic immunoprecipitation test.