Neurospora tyrosinase: structural,spectroscopic and catalytic properties

Summary Tyrosinase is a copper containing monooxygenase catalyzing the formation of melanin pigments and other polyphenolic compounds from various phenols. This review deals with the recent progress on the molecular structure of the enzyme from Neurospora crassa and the unique features of the binuclear active site copper complex involved in the activation of molecular oxygen and the binding of substrates. The results of the spectroscopic properties of Neurospora tyrosinase will also be discussed in the light of the structural similarity of the copper complex in the oxygen binding hemocyanins.     

Cyclic AMP-induced tyrosinase synthesis in Neurospora crassa

Cyclic AMP induces the synthesis of tyrosinase in $\text{Neurospora crassa}$. Adenine, adenosine, 3′-AMP, 5′-AMP, and 2′,3′-cyclic AMP have no inductive effect while 8-bromocyclic AMP and dibutyryl cyclic AMP are good inducers. Caffeine and theophylline, inhibitors of cyclic AMP phosphodiesterase, also induce tyrosinase. A possible relationship between cyclic AMP induction and previously reported induction by cycloheximide is suggested.     

Cobalt tyrosinase: replacement of the binuclear copper of Neurospora tyrosinase by cobalt

The antiferromagnetically spin-coupled copper(II) pair in Neurospora tyrosinase was substituted by cobalt, yielding a stoichiometry of 2 mol of Co/mol of protein. The low magnitude of the high-spin Co(II) EPR signal indicates spin coupling of the two Co(II) ions similar to that observed in the native enzyme. The absorption spectrum with four transitions in the visible region of intermediate intensity (epsilon 607(670), epsilon 564(630), epsilon 526(465)), a shoulder at 635 nm, and the near-infrared bands at 1180 (epsilon 30) and 960 nm (epsilon 15) indicate tetrahedral coordination around the Co(II) center. The cobalt(II) tyrosinase is enzymatically inactive, and there is no evidence that it binds molecular oxygen. Upon addition of cyanide or the competitive tyrosinase inhibitors L-mimosine, benzoic acid, or benzhydroxamic acid te absorption spectrum changes in a characteristic manner. This optical perturbation shows that binding of these inhibitors (and presumably of the substrates) occurs at or near the metal site. One Co(II) ion can be removed preferentially by incubation with KCN at high pH, indicating the two ions not to be in an identical environment.     

Histidine at the active site of Neurospora tyrosinase

The involvement of histidyl residues as potential ligands to the binuclear active-site copper of Neurospora tyrosinase was explored by dye-sensitized photooxidation. The enzymatic activity of the holoenzyme was shown to be unaffected by exposure to light in the presence of methylene blue; however, irradiation of the apoenzyme under the same conditions led to a progressive loss of its ability to be reactivated with Cu2+. This photoinactivation was paralleled by a decrease in the histidine content whereas the number of histidyl residues in the holoenzyme remained constant. Copper measurements of photooxidized, reconstituted apoenzyme demonstrated the loss of binding of one copper atom per mole of enzyme as a consequence of photosensitized oxidation of three out of nine histidine residues. Their sequence positions were determined by a comparison of the relative yields of the histidine containing peptides of photooxidized holo- and apotyrosinases. The data obtained show the preferential modification of histidyl residues 188, 193, and 289 and suggest that they constitute metal ligands to one of the two active-site copper atoms. Substitution of copper by cobalt was found to afford complete protection of the histidyl residues from being modified by dye-sensitized photooxidation.     

A rapid and efficient method for the purification of tyrosinase from Neurospora.

A short and efficient procedure consisting of two chromatographic steps is described for the isolation of tyrosinase from Neurospora. The first step, Celite-column chromatography, resulted in the isolation of four copper-containing proteins from the crude mycelial extract. Anion-exchange chromatography on DEAE-Sephadex of these proteins resulted in the isolation of electrophoretically and serologically pure tyrosinase. More than 70% of the initial tyrosinase activity was recovered in the final enzyme preparation, which had a specific activity of 450 units/mg.     

Isolation and characterization of the tyrosinase gene from Neurospora crassa

A precursor form of Neurospora crassa tyrosinase has been identified by Western transfer from crude protein extracts and by immunoprecipitation of in vitro translated tyrosinase mRNA. The molecular weight of protyrosinase (75,000) exceeds that of mature tyrosinase (46,000) by about 50%. In order to deduce the primary structure and the nature of the extension, the tyrosinase gene was cloned. Poly(A) RNA isolated from tyrosinase-induced cultures of N. crassa was used as a template for cDNA synthesis, primed by a tyrosinase-specific, 32-fold degenerate heptadecanucleotide. Based on this sequence, a unique 21-mer was synthesized and used to screen a cDNA library constructed from tyrosinase-enriched mRNA. A partial genomic DNA library from wild-type strain TS and a genomic library from strain OR were screened using a 400-base pair nick-translated SalI fragment from a tyrosinase-positive cDNA clone as hybridization probe. The DNA sequences obtained revealed the presence of two allelic forms of this enzyme. The coding regions are interrupted by two short introns, of 52 and 99 base pairs. The encoded proteins differ in 3 out of 621 amino acid residues. A comparison of the deduced amino acid sequence with the known primary structure of mature tyrosinase alleles (Rüegg, C., Ammer, D., and Lerch, K. (1982) J. Biol. Chem. 257, 6420-6426) showed that the enzyme is synthesized as a precursor. Protyrosinase exceeds the mature protein by 213 amino acids at its carboxyl terminus. The possible involvement of carboxyl-terminal processing in enzyme activation is discussed.     

Optimization of simultaneous production of tyrosinase and laccase by Neurospora crassa

Increasing demand for efficient and environmentally benign oxidation technologies has resulted in a focus on the use of oxidoreductases. Laccases and tyrosinases, which utilize molecular oxygen and produce water as by-product, are particularly attractive. Simultaneous production of laccase and tyrosinase was studied in Neurospora crassa FGSC #321 as the fungal strain which has the ability to produce tyrosinase intracellularly while producing laccase extracellularly. Using one-variable-at-a-time experiments and a Taguchi orthogonal L9 array demonstrated that a Vogel minimal medium containing 2.5% sucrose at pH 6.5 and 25?°C with no agitation or oxygen purging were the optimum conditions for N. crassa FGSC #321 growth. Conditions were adjusted to obtain the highest laccase and tyrosinase production. Results indicate that the control mechanisms for the production of both enzymes in N. crassa FGSC #321 are similar but not necessarily identical. Results revealed that transferring the harvested cells from the growth medium into the phosphate buffer (pH 6.8, 0.1M) containing cycloheximide (2?μM) and fluorouracil (2?mM) and increasing the temperature to 30?°C were the best conditions for simultaneous production of laccase and tyrosinase (1278 and 410?U/g of biomass, respectively). Nonetheless, starvation at 35?°C is proposed as the most cost-effective means for inducing laccase. The N. crassa laccase was characterized by using its molecular weight, pI value, optimal pH and temperature and stability.     

Hydrodynamic properties of mushroom tyrosinase

The hydrodynamic properties of mushroom tyrosinase were determined at pH 6.5 using a Sephadex G-200 column. From the comparison of its gel-filtration behaviour with those of standard proteins, the following parameters were calculated: MW (122 500 ± 1%), Stokes' radius (42.75 × 10^?8 cm²/sec), diffusion coefficient (5.048 × 10^?7 cm²/sec) and frictional ratio (1.26). These values suggest a globular conformation of this enzyme.     

Fluorescence properties of terbium-alkaline phosphatase.

The addition of Tb³⁺ to apoalkaline phosphatase at pH 8.0 results in the formation of a metalloprotein with an enhanced Tb³⁺ fluorescence at 492, 545, and 580 nm. The Tb³⁺ excitation spectrum is most consistent with a process in which energy is transferred from one or more tyrosyl chromophores to the bound lanthanide. An analysis of the fluorescence data under equilibrium conditions yields one Tb³⁺ binding site per enzyme dimer with a K_n = 0.16 ± 0.02 μm. The Tb³⁺-alkaline phosphatase complex is not catalytically active nor does it incorporate covalently bound phosphate, but the specific activity of Zn²⁺-alkaline phosphatase is significantly enhanced in the presence of Tb³⁺ indicating that this lanthanide mimics Mg²⁺ in stabilizing the structure of alkaline phosphatase. The fluorescence of the Tb³⁺-enzyme is found to be quite sensitive to conformational changes which occur upon addition of Zn²⁺ or substrates.     

Regulation of tyrosinase during the vegetative and sexual life cycles of Neurospora crassa

Tyrosinase derepression in Neurospora mycelia grown in Vogel medium, submitted to starvation in phosphate buffer 0.1 M, pH 6.0, was abolished by exogenous magnesium sulfate. This effect seemed to be caused by the sulfate ion itself and not by a sulfate-derivative. Sulfate repression required protein synthesis, thus suggesting the involvement of a specific gene product mediating sulfate repression. Cultures made in Westergaard and Mitchell crossing medium became competent for sexual development and could be stimulated to form tyrosinase either by mating or starvation. In that case the enzyme derepression was insensitive to the sulfate effect. The possible existence of a positive mechanism for the control of tyrosinase activity during sexual development is suggested.This work is a part of two theses, by Rolf Alexander Prade and Angela Kaysel Cruz submitted to the Departments of Biochemistry and Physiology, respectively, of the School of Medicine of Ribeirão Preto in partial fulfillments of the requirements for the Master Degree.     

Role of sulfhydryl compounds in the control of tyrosinase activity in Neurospora crassa

It is known that Neurospora crassa mycelia cultured in standard concentrations (76 to 190 µg/ml) of sulfate accumulate a low molecular weight inhibitor of tyrosinase (monophenol, dihydroxyphenylalanine: oxygen oxidorenductase; EC 1.14.1.18.1.). This is not observed in cultures grown under sulfate-limiting conditions. The chemical nature of tyrosinase inhibition was investigated. It was shown to be due to the low molecular weight sulfhydryl fraction of the extracts, in which glutathione is predominant. The concentration of low molecular weight sulfhydryl compounds decreased sharply in mycelia submitted to various treatments which also derepressed tyrosinase, such as (i) starvation in phosphate buffer, (ii) treatment with cycloheximide, and (iii) mating. These results suggest that the concentration of sulfhydryl compounds may be of physiological significance in the control of tyrosinase activity in N. crassa.