Neurospora Tyrosinase Research Articles

Use of the Neurospora tyrosinase gene as a reporter gene in transformation experiments

Use of the Neurospora tyrosinase gene as a reporter gene in transformation experiments

The reaction of CN − with the binuclear copper site of Neurospora tyrosinase: its relevance for a comparison between tyrosinase and hemocyanin active sites

The equilibrium and the kinetics of the reaction between Neurospora crassa tyrosinase and cyanide have been studied. Cyanide reacts with the binuclear copper active site of the protein competitively with respect to dioxygen and displaces the metal ions. This process occurs stepwise and involves transient intermediates containing mononuclear Cu(I) sites. The reaction mechanism proved to be the same as described earlier for molluscan and arthropodan hemocyanins, which share with tyrosinase the same copper active site organization, but perform different physiological functions. A comparison of the kinetic parameters between the different proteins shows that the tyrosinase copper site has a greater accessibility than that of hemocyanin. The relevance of these data in terms of structure-function relationship and evolution of the binuclear copper proteins is discussed.

Cloning and expression of cDNA encoding mouse tyrosinase.

We have isolated a pigment cell-specific cDNA clone from a B16 mouse melanoma cDNA library by differential hybridization. The mRNA of isolated cDNA is highly expressed in B16 melanoma cells and in black mouse (C57BL/6) skin, but is not detectable in mouse neuroblastoma cells nor in K1735 mouse amelanotic melanoma cells. The protein sequence deduced from the nucleotide sequence of the cloned cDNA shows significant similarity to the entire region of Neurospora tyrosinase. To know the identity of cDNA, we transfected K1735 amelanotic melanoma and COS-7 cells with the cDNA carried in a simian virus 40 vector (pKCRH2). We confirmed that the isolated cDNA encodes mouse tyrosinase by immunofluorescence staining of transfected cells using two different anti-T4-tyrosinase monoclonal antibodies. Tyrosinase is composed of 513 amino acids with a molecular weight of 57,872 excluding a hydrophobic signal peptide of 24 amino acids.

35] Sβ-(2-histidyl)cysteine: Properties, assay, and occurrence

Publisher Summary This chapter discusses the physicochemical properties of S β -(2-Histidyl)cysteine. It also describes an analytical procedure for its detection in peptides and proteins. The chemical structure of the tripeptide Cys-Thr-His [residues 94-96 of Neurospora tyrosinase containing S β -(2-Histidyl)cysteine] is characterized by an intramolecular thioether linking the S β - cysteine sulfur to the C-2 imidazole carbon of the histidine moiety. The presence of a thioether in this peptide is also substantiated by a positive chloroplatinic acid test and by the cleavage with Ag 2 SO 4 resulting in cysteic acid after performic acid oxidation. The determination of S β -(2-Histidyl)cysteine is based on the diagonal procedure of Brown and Hartley using ion-exchange chromatography. The first step involves cleavage of the thioether by acid hydrolysis and subsequent isolation of 2-thiolhistidine. In a second step, 2-thiolhistidine is converted to histidine with hydrogen peroxide. Histidine is then quantitated by amino acid analysis. This procedure is applicable to any protein or peptide supposedly containing this thioether structure. S β -(2-Histidyl)cysteine is detected in three different allelic forms of Neurospora tyrosinase.

Photoinactivation of agaricus bisporus tyrosinase: modification of the binuclear copper site.

Irradiation of Agaricus bisporus tyrosinase in the presence of citrate at pH 5.6 with 300-420-nm light results in a loss of both catecholase activity and cresolase activity. The light-sensitive species appears to be an enzyme-citrate complex, most likely involving coordination of citrate to the active site copper. One copper ion from each binuclear active site can be removed from the inactivated enzyme, resulting in the formation of a met apo derivative. The electron spin resonance spectrum of met apo tyrosinase resembles that of met apo hemocyanin and half-met Neurospora tyrosinase. It is consistent with a distorted square-planar geometry around the copper and with either nitrogen or nitrogen and oxygen ligands. Amino acid analysis indicates that four histidines on the heavy subunit are destroyed during the inactivation process. Some or all of these histidines may serve as ligands to the copper ion which becomes labile after inactivation. Photoinactivation results in decarboxylation of citrate and does not require the presence of oxygen. The reaction may involve generation of a free radical from the citrate which then attacks nearby histidine residues.

Neurospora tyrosinase: structural, spectroscopic and catalytic properties.

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.

Fluorescence properties of Neurospora tyrosinase.

Some structural properties of Neurospora tyrosinase have been studied by fluorescence spectroscopy. The emission spectra observed for oxy-, deoxy-, met- and apo-tyrosinase and the Co2+-substituted form are indicative of a protein containing buried tryptophan residues. By using acrylamide and iodide, part of the emission is quenched, indicating heterogeneity in the tryptophan environment. Upon binding of Cu2+ or Co2+ to apo-tyrosinase, a marked decrease of the tryptophan quantum yield is observed. A further decrease in emission intensity results from the binding of molecular O2 to the deoxy form. The fluorescent probe 8-anilinonaphthalene-1-sulphonate binds to tyrosinase only when the metal ions are removed. Reconstitution of apo-tyrosinase with Cu2+ completely displaces the probe, suggesting that 8-anilinonaphthalene-1-sulphonate binds to apo-tyrosinase at the active site. The fluorescence properties of Neurospora tyrosinase are compared with those of haemocyanin.

Primary structure of tyrosinase from Neurospora crassa. II. Complete amino acid sequence and chemical structure of a tripeptide containing an unusual thioether.

To align the four cyanogen bromide peptides of Neurospora tyrosinase whose amino acid sequences were reported in the preceding paper, suitable methionine-containing overlap peptides were isolated. The required peptides were obtained by tryptic, peptic, and thermolytic digestion of the unmodified protein and of the maleylated derivative. From the partial sequence information of these peptides and a cyanogen bromide overlap peptide, the four cyanogen bromide fragments were aligned in the order CB3-CB1-CB4-CB2. These data establish Neurospora tyrosinase as a single-chain protein of 407 amino acids with a molecular weight of 46,000. The single cysteinyl residue 94 was found to be covalently linked via a thioether bridge to histidyl residue 96. The chemical nature of this unusual structure was elucidated by physicochemical analysis of peptides obtained from in vivo 35S, [2,5-3H]histidine, and [5-3H]histidine-labeled Neurospora tyrosinase.

Primary structure of tyrosinase from Neurospora crassa. I. Purification and amino acid sequence of the cyanogen bromide fragments.

Cyanogen bromide (CB) cleavage of Neurospora tyrosinase resulted in four major fragments, CB1 (222 residues), CB2 (82 residues), CB3 (68 residues), and CB4 (35 residues), and one minor overlap peptide CB2-4 (117 residues) due to incomplete cleavage of a methionylthreonyl bond. The sum of the amino acid residues of the four major fragments matches the total number of amino acid residues of the native protein. The amino acid sequences of the cyanogen bromide fragments CB2, CB3, and CB4 were determined by a combination of automated and manual sequence analysis on peptides derived by chemical and enzymatic cleavage of the intact and the maleylated derivatives. The peptides were the products of cleavage by mild acid hydrolysis, trypsin, pepsin, chymotrypsin, thermolysin, and Staphylococcus aureus protease V8. The cyanogen bromide fragment CB1 was found to contain two unusual amino acids whose chemical structure will be presented in the following paper.

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.

Pulsed EPR studies of peroxide-activated cytochrome c peroxidase and of the mercaptoethanol derivative of Neurospora tyrosinase.

We have measured the linear electric field effect for peroxide-activated yeast cytochrome c peroxidase and for the mercaptoethanol derivative of Neurospora tyrosinase. Although both of these materials have EPR spectra resembling those of free radicals, the linear electric field effect measurements demonstrate that there is a metal ion associated with the paramagnetic centers. In addition, we have also observed superhyperfine interactions with 14N nuclei.

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.

Polypeptide composition of two fungal tyrosinases

The presence of two distinct molecular structures for tyrosinase in fungi is confirmed. The enzyme from Agaricus bisporus is acidic and comprises two dissimilar subunits which aggregate to form a tetramer. This tetramer constitutes the majority both in the resting and functional states. In Neurospora crassa, tyrosinase is slightly basic and contains only one subunit, similar in size to the larger subunit of the Agaricus enzyme. In the resting state Neurospora tyrosinase is distributed among a number of forms, from the monomer to the tetramer. In this case it was possible to show that a species smaller than the tetramer, probably the monomer, was fully active.

Chemical and spectroscopic studies of the binuclear copper active site of Neurospora tyrosinase: comparison to hemocyanins

Chemical and spectroscopic studies of the binuclear copper active site of Neurospora tyrosinase: comparison to hemocyanins

Luminescence of the copper—carbon monoxide complex of Neurospora tyrosinase

Luminescence of the copper—carbon monoxide complex of Neurospora tyrosinase

Metal substitution of neurospora tyrosinase

The antiferromagnetically spin coupled copper pair of Neurospora tyrosinase [1] was substituted with cobalt and cadmium. Cobalt is bound specifically at the copper-binding site with a stoichiometry of 2 Co(II) per mole protein. The visible and near infrared absorption spectrum of this derivative (ϵ 526 = 465, ϵ 564 = 630, ϵ 670 = 670, ϵ 635 = 460 (sh), hedral coordination of the cobalt chromophore. An estimation of the ligand field strength (∼5300 cm −1) indicates nitrogen ligation (presumably imidazole side chains) which is in accordance with protein chemical modification data [2]. The low intensity of the Co EPR signal (high spin Co(II)) suggests similar anti-ferromagnetic coupling of the metal ions as in the native enzyme. Contrary to low molecular weight, binuclear Cocomplexes, Cotyrosinase does not bind molecular oxygen. This finding can be explained by the apparent constraint of the Co-chromophore to tetrahedral coordination in the protein, whereas all binuclear cobalt oxygen complexes are octahedral [3]. Competitive and noncompetitive tyrosinase inhibitors (benzoic acid, L-mimosine and KCN) give rise to characteristic perturbations of the absorption spectrum of Cotyrosinase. The spectral changes are taken as evidence for the direct interaction of the inhibitors with the metal site. Cadmium substitution of native tyrosinase leads to an enzymatically inactive derivative containing only one Cd(II) per mole protein. A Cu/Cd hybrid form is obtained upon addition of stoichiometric amounts of Cu(II); excess copper expells the cadmium from the hybrid yielding native tyrosinase.

The reaction of mercaptans with tyrosinases and hemocyanins

1. 1. Titration of Neurospora tyrosinase with 2-mercaptoethanol shows that the increase of absorbance at 700 nm is directly correlated to the loss of enzymatic activity. Approximately 2 mol of 2-mercaptoethanol per mole of protein are needed for full development of the green, enzymatically inactive complex. The increase of absorbance at 700 nm is also proportional to the intensity of the EPR signal and the amount of non-covalently bound 2-[ 35S]mercaptoethanol to the enzyme. The maximal EPR intensity reaches 70% of the protein concentration and at most 0.7–0.8 mol of 2-[ 35S]mercaptoethanol is bound per mol of enzyme. 2. 2. Stopped-flow measurements show that in the reaction between 2-mercaptoethanol and Neurospora tyrosinase a reaction intermediate with a strong absorption band at 360 nm is formed in an apparent second-order reaction. This intermediate displays no EPR-detectable signals. The intermediate decays in a similar complex fashion as the absorption band at 700 nm is formed. 3. 3. The reaction of Neurospora tyrosinase with a variety of sulfhydryl compounds was also investigated. In most cases green coloured, enzymatically inactive complexes are formed displaying slightly different EPR signals. However, with cysteine and cysteamine violet coloured, enzymatically inactive complexes are formed which show rather different EPR signals. The integrated EPR intensities amount to 40–70% of the protein concentration. Based on simulations of 9 and 35 GHz spectra all observed EPR spectra can be represented as true S = 1 2 systems. The cysteamine complex can be interpreted as arising from a mixed valence Cu 2+ · Cu + complex. The 2-mercaptoethanol spectra can, however, arise from sulphur radicals. 4. 4. Treatment of Agaricus bispora tyrosinase and Cancer pagures hemocyanin with 2-mercaptoethanol results in green-coloured, EPR detectable complexes similar to the one found with Neurospora tyrosinase. No such complexes are formed when hemocyanins from Helix pomatia and Panulirus interruptus were treated with this reagent.

Amino acid sequence of tyrosinase from Neurospora crassa.

The amino-acid sequence of tyrosinase from Neurospora crassa (monophenol,dihydroxyphenylalanine:oxygen oxidoreductase, EC 1.14.18.1) is reported. This copper-containing oxidase consists of a single polypeptide chain of 407 amino acids. The primary structure was determined by automated and manual sequence analysis on fragments produced by cleavage with cyanogen bromide and on peptides obtained by digestion with trypsin, pepsin, thermolysin, or chymotrypsin. The amino terminus of the protein is acetylated and the single cysteinyl residue 96 is covalently linked via a thioether bridge to histidyl residue 94. The formation and the possible role of this unusual structure in Neurospora tyrosinase is discussed. Dye-sensitized photooxidation of apotyrosinase and active-site-directed inactivation of the native enzyme indicate the possible involvement of histidyl residues 188, 192, 289, and 305 or 306 as ligands to the active-site copper as well as in the catalytic mechanism of this monooxygenase.

Amino acid sequence determination of the blocked N-terminal tryptic peptide of Neurospora tyrosinase by mass spectrometry

FEBS LettersVolume 79, Issue 1 p. 203-206 Full-length articleFree Access Amino acid sequence determination of the blocked N-terminal tryptic peptide of Neurospora tyrosinase by mass spectrometry Heinz Nau, Corresponding Author Heinz Nau Institut für Toxikologie und Embryonalpharmakologie der Freien Universität, Garystraße 9, D-1000 Berlin 33, FRG Biochemisches Institut der Universität Zürich, CH-8028 Zürich, Switzerland Gesellschaft für Biotechnologische Forschung, D-3300 Stöckheim/Braunschweig, FRGTo whom correspondence should be addressedSearch for more papers by this author Heinz Nau, Corresponding Author Heinz Nau Institut für Toxikologie und Embryonalpharmakologie der Freien Universität, Garystraße 9, D-1000 Berlin 33, FRG Biochemisches Institut der Universität Zürich, CH-8028 Zürich, Switzerland Gesellschaft für Biotechnologische Forschung, D-3300 Stöckheim/Braunschweig, FRGTo whom correspondence should be addressedSearch for more papers by this author First published: July 01, 1977 https://doi.org/10.1016/0014-5793(77)80384-0Citations: 9AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 W.H. Vanneste, A. Zuberbühler, O. Hayaishi Molecular Mechanisms of Oxygen Activation (1974), Academic Press New York 2 K. Lerch, FEBS Lett., 69, (1976), 157– 160. 3 K. Okada, S. Nagai, H. Kato, Experientia, 30, (1974), 459– 460. 4 G.M. Hass, H. Nau, K. Biemann, D.T. Grahn, L.H. Ericsson, H. Neurath, Biochemistry, 14, (1975), 1334– 1342. 5 R.L. Jolley, R.M. Nelson, D.A. Robb, J. Biol. Chem., 244, (1969), 3251– 3257. 6 K.G. Strothkamp, R.L. Jolley, H.S. Mason, Biochem. Biophys. Res. Commun., 70, (1976), 519– 523. 7 K. Lerch, 10th Meeting Federation of European Biochemical Societies, Paris, France., (1975), 8 H. Baumann, K.J. Wilson, P.S. Chen, R.E. Humbel, Eur. J. Biochem., 52, (1975), 521– 529. 9 R.L. Hill, R. Delaney, Meth. Enzymol., 11, (1967), 339– 351. 10 W.J. Dreyer, E. Bynum, Meth. Enzymol., 11, (1967), 32– 39. 11 H. Fraenkel-Conrat, C.M. Tsung, Meth. Enzymol., 11, (1967), 151– 155. 12 P.A. Leclercq, D.M. Desiderio, Anal. Lett., 4, (1971), 305– 316. 13 H.R. Morris, FEBS Lett., 22, (1972), 257– 260. 14 R.A. Hites, K. Biemann, Anal. Chem., 42, (1970), 855– 860. 15 K. Biemann, F. Gapp, J. Seibl, J. Amer. Chem. Soc., 81, (1959), 2274– 2276. 16 J.A. Kelley, H. Nau, J.A. Förster, K. Biemann, Biomed. Mass Spectrom., 2, (1975), 313– 325. 17 H. Nau, Biochem. Biophys. Res. Commun., 59, (1974), 1088– 1096. 18 H.R. Morris, D.H. Williams, R.P. Ambler, Biochem. J., 125, (1971), 189– 201. 19 H. Nau, J.A. Kelley, H.-J. Förster, K. Biemann, Biomed. Mass Spectrom., 2, (1975), 326– 339. Citing Literature Volume79, Issue1July 01, 1977Pages 203-206 ReferencesRelatedInformation

Neurospora tyrosinase: Molecular weight, copper content and spectral properties

Neurospora tyrosinase: Molecular weight, copper content and spectral properties