Protein and active-site structure of tyrosinase.
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Amino Acid Sequence↗
Biomedical subjects
K Lerch
Publications and source records attributed to K Lerch.
Evolutionary relationships among copper proteins containing coupled binuclear copper sites.
The amino acid sequences of different copper proteins containing coupled binuclear copper centers are compared. Hemocyanins from arthropods and molluscs and tyrosinases from three different species were found to share a highly homologous region in the C-terminal parts. This region contains three invariant histidines previously identified as ligands to Cu(B) in Panulirus interruptus hemocyanin by X-ray crystallography (Gaykema et al., Nature 309, 23-29 (1984]. In contrast, the ligand environment for the second copper, Cu(A), proved to be quite variable. It is proposed that hemocyanin and tyrosinase have arisen from a common mononuclear copper protein with the typical Cu(B) site. From this ancestral protein two types of binuclear proteins evolved independently into a tyrosinase and an arthropodan hemocyanin type. The amino acid sequence comparison between human ceruloplasmin and Neurospora crassa laccase together with the results from a preliminary X-ray structure analysis of zucchini ascorbate oxidase showed a close relationship in primary and most likely also in tertiary structure in the C-terminal parts of these enzymes. It is suggested that the multicopper oxidases have evolved from an ancestral copper protein which presumably contained all the ligands required for the binding of one binuclear and two additional mononuclear metal centers.
Animals↗
Catalytic oxidation of 2-aminophenols and ortho hydroxylation of aromatic amines by tyrosinase.
The usual substrates of tyrosinase, a copper-containing monooxygenase (EC 1.14.18.1), are monophenols and o-diphenols which are both converted to o-quinones. In this paper, we studied the reaction of this enzyme with two new classes of substrates: aromatic amines and o-aminophenols, structural analogues of monophenols and o-diphenols, respectively. They undergo the same catalytic reactions (ortho hydroxylation and oxidation), as documented by product analysis and kinetic studies. In the presence of tyrosinase, arylamines and o-aminophenols are converted to o-quinone imines, which are isolated as quinone anils or phenoxazones. As an example, in the presence of tyrosinase, 2-amino-3-hydroxybenzoic acid (an o-aminophenol) is converted to cinnabarinic acid, a well-known phenoxazone, while p-aminotoluene (an aromatic amine) gives rise to the formation of 5-amino-2-methyl-1,4-benzoquinone 1-(4-methylanil). Kinetic studies using an oxygen electrode show that arylamines and the corresponding monophenols exhibit similar Michaelis constants (Km = 0.11-0.49 mM). In contrast, the reaction rates observed for aromatic amines are relatively slow (Kcat = 1-3 min-1) as compared to monophenols (1320-6960 min-1). The enzymatic conversion of arylamines by tyrosinase is different from the typical ones: N-oxidation and ring hydroxylation without further oxidation. This difference originates from the regiospecific hydroxylation (ortho position) and subsequent oxidation of the intermediate o-aminophenol to the corresponding o-quinone imine. Finally, the well-known monooxygenase activity of tyrosinase was also confirmed for the aromatic amine p-aminotoluene, with 18O2.(ABSTRACT TRUNCATED AT 250 WORDS)
Amines↗
The promoter of the Streptomyces glaucescens mel operon.
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Amino Acid Sequence↗
The influence of copper on the induction of tyrosinase and laccase in Neurospora crassa.
The influence of copper on the cycloheximide-induced synthesis of the copper-containing enzymes tyrosinase and laccase in Neurospora crassa was studied by enzyme activity measurements and immunological means. The amount of active enzyme molecules is far higher when the culture medium is copper-supplemented before cycloheximide induction. The synthesis of the apoproteins is not dependent on the presence of copper. This suggests the existence of a copper-storage protein for which metallothionein is a likely candidate.
Catechol Oxidase↗
Copper accumulation in the cell-wall-deficient slime variant of Neurospora crassa. Comparison with a wild-type strain.
The copper-uptake process in the cell-wall-deficient slime variant of the fungus Neurospora crassa was compared with that in a wild-type strain. In both organisms investigated most of the copper is taken up from the culture medium during the exponential growth period. The wild-type strain, however, accumulates much more copper than does the slime variant. The influence of the copper concentration in the culture medium on the amounts of copper accumulated intracellularly suggests separate ways of copper import used by the two morphologically different N. crassa strains. Copper analyses of three different cytosolic fractions as a function of growth time or exogenous copper concentration indicate both strains to share a very similar copper metabolism. All the data presented are consistent with a detoxification function of the low-Mr copper-binding fraction of N. crassa. Both copper-metallothionein and oxidized glutathione (GSSG) are co-eluted with this fraction. The possible involvement of glutathione in metallothionein biosynthesis is discussed.
Amino Acids↗
The Neurospora crassa metallothionein gene. Regulation of expression and chromosomal location.
The promoter region of the Neurospora crassa metallothionein gene contains no sequences which are similar to the mammalian or the yeast metal responsive elements (Münger, K., Germann, U. A., and Lerch, K. (1985) EMBO J. 4, 2665-2668). We therefore studied the regulation of expression of the N. crassa metallothionein gene in response to different metal ions (Cu2+, Cd2+, Zn2+, Co2+, and Ni2+) by Northern analysis. Only copper led to the induction of metallothionein mRNA. In N. crassa cultures inoculated and grown in copper-supplemented media, metallothionein mRNA appeared during the late logarithmic growth period (about 30 h after inoculation) and was detectable for a time period of more than 30 h. In response to copper shock, however, rapidly increasing amounts of metallothionein mRNA were detected within minutes after copper administration at any time in vegetatively growing mycelia of N. crassa. Maximum levels were detected about 1 h after addition of copper to the medium. The half-life time of the mRNA was estimated as 2.5 h. The amounts of copper metallothionein reach a maximum level at 3 h after induction and thereafter remain constant. The rapid induction by copper ions of metallothionein mRNA and metallothionein together with the remarkable stability of the native protein intracellularly suggest that this protein serves an important homeostatic role in the copper metabolism in this fungus. The structural gene of N. crassa metallothionein has been located on chromosome VI using restriction fragment-length polymorphisms as genetic markers.
Cations, Divalent↗
Luminescence emission from the Cu(I)-thiolate complex in metallothioneins.
The luminescence emission properties of Cu-metallothioneins (from Neurospora crassa, Agaricus bisporus and livers of Bedlington terriers affected by copper toxicosis) as well as of (Cu,Zn)-metallothionein from bovine fetal liver are reported. Upon excitation in the U.V., these proteins emit a largely red-shifted luminescence with a maximum at 565 nm attributable to the Cu(I)-thiolate chromophores of the proteins. Differences in the shapes of the spectra and the emission intensity are observed with (Cu,Zn)-metallothionein probably due to the influence of Zn ions or to a different coordination geometry of the Cu ions. The emissive properties of the Cu(I)-thiolate chromophore are compared with those of metallo-organic Cu(I)-mercaptide complexes.
Agaricus↗
Isolation and regulation of expression of the Neurospora crassa copper metallothionein gene.
The N. crassa CuMT gene has been cloned and its nucleotide sequence determined. To this end an MT specific undecanucleotide was synthesized and used for cDNA synthesis with enriched MT mRNA as a template. Sequence analysis of the cDNA obtained allowed the synthesis of a unique 21mer which was used as a hybridization probe to screen a genomic DNA library of N. crassa. Several positive clones were isolated and subjected to restriction and sequence analysis. In agreement with the published amino acid sequence, the gene codes for a polypeptide of 26 amino acid residues in length. The coding region is interrupted by a small intron. Compared to the structure of mammalian MT genes the intron-exon boundaries are located in different sequence positions. The induction of MT mRNA was studied by Northern analysis. Maximum levels of MT mRNA were detected about 1 hour after addition of copper ions to mycelium of N. crassa. The half-life time of the messenger was estimated as 2.5 hours. The CuMT amounts reach a maximum level at 3 hours after induction and thereafter remain constant.
Amino Acid Sequence↗
Monophenol monooxygenase from Neurospora crassa.
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Catechol Oxidase↗
Isolation and partial nucleotide sequence of the laccase gene from Neurospora crassa: amino acid sequence homology of the protein to human ceruloplasmin.
The laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) gene from Neurospora crassa was cloned and part of its nucleotide sequence corresponding to the carboxyl-terminal region of the protein has been determined. The gene was cloned by cDNA synthesis with a laccase-specific synthetic deoxyundecanucleotide as primer and poly(A) RNA isolated from cycloheximide-treated N. crassa cultures as template. Based on the nucleotide sequence of the cDNA obtained, a unique 21-mer was synthesized and used to screen a genomic DNA library from N. crassa. Five different positive clones were isolated and shown to share an overlapping DNA region with the same pattern of restriction sites. Sequence analysis of the common 1.36-kilobase Sal I fragment revealed an open reading frame of 726 nucleotides. The amino acid sequence deduced is in complete agreement with the primary structures of several tryptic peptides isolated previously from N. crassa laccase. The analyzed carboxyl-terminal region of laccase exhibits a striking sequence homology to the carboxyl-terminal part of the third homology unit of the multicopper oxidase ceruloplasmin and to a smaller extent, to the low molecular weight blue copper proteins plastocyanin and azurin. Based on amino acid sequence comparison between these proteins, putative copper ligands of N. crassa laccase are proposed. Moreover, these data further support the hypothesis that the small blue copper proteins and the multicopper oxidases have evolved from the same ancestral gene.
Amino Acid Sequence↗
Primary structure and spectroscopic studies of Neurospora copper metallothionein.
When Neurospora crassa is grown in the presence of Cu(II) ions, it accumulates the metal with the concomitant synthesis of a low molecular weight copper-binding protein. The molecule binds 6 g-atom of copper per mole protein (Mr = 2200) and shows a striking sequence homology to the zinc- and cadmium-binding vertebrate metallothioneins. Absorption, circular dichroism, and electron paramagnetic resonance spectroscopy of Neurospora metallothionein indicate the copper to be bound to cysteinyl residues as a Cu(I)-thiolate complex of the polymeric mu-thiolate structure [Cu(I)6RS7]-. This metal-binding mode is also in agreement with the unusual luminescence of the protein. Spectral perturbation studies with HgCl2 and p-(chloromercuri)benzoate suggest that the 6 Cu(I)ions are coordinated to the seven cysteinyl residues in the form of a single metal cluster. Neurospora apometallothionein is also capable of binding in vivo group IIB metal ions [Zn(II), Cd(II), and Hg(II)] as well as paramagnetic Co(II) ions with an overall metal-to-protein stoichiometry of 3. The spectroscopic properties of the fully substituted forms are indicative of a distorted tetrahedral coordination. However, metal titration of the apoprotein shows the third metal ion to be differently coordinated than the other two metal ions. This difference can be explained by the presence of only seven cysteine residues in Neurospora metallothionein as opposed to nine cysteine residues in the three-metal cluster of the mammalian metallothioneins.
Amino Acid Sequence↗
[Results of treating fetal hemolytic disease using intrauterine transfusions].
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Blood Transfusion, Intrauterine↗
Canine hepatic lysosomal copper protein: identification as metallothionein.
We studied the amino acid sequence of canine hepatic lysosomal copper protein obtained from Bedlington terriers affected by inherited copper toxicosis. The primary structure was determined by manual Edman degradations and carboxypeptidase Y digestions of peptides generated by cleavage of the S-carboxyamidomethylated and S-aminoethylated protein with trypsin. Although the amino terminus was blocked and heterogeneous, the protein showed extensive sequence homology to mammalian metallothioneins. In particular, all cysteinyl residues were conserved, in agreement with their function as metal ligands. The microheterogeneity observed in the amino-terminal part of the molecule indicated the presence of two isoforms in canine liver like those found in most other mammals studied so far.
Amino Acid Sequence↗
Primary structure of tyrosinase from Streptomyces glaucescens.
The complete amino acid sequence of Streptomyces glaucescens tyrosinase is reported. The molecule consists of 273 amino acids and has a Mr of 30 900 including two copper atoms. The primary structure was determined by a combination of amino acid and DNA sequence analysis. Peptide sequence information was derived from the cyanogen bromide, tryptic, and thermolytic fragments of apotyrosinase by automated Edman degradation and aminopeptidase M and carboxypeptidase C digestions. The nucleotide sequence of the tyrosinase gene cloned into the PvuII site of pBR322 was determined. The enzyme contains no apparent leader peptide despite the fact that it is secreted into the culture medium. As observed for a number of different Streptomyces genes, the tyrosinase gene shows a strong preference (97%) for codons ending in G or C. A comparison of the amino acid sequence of Streptomyces glaucescens tyrosinase with that of Neurospora crassa tyrosinase reveals an overall sequence homology of only 24.2%. However, the sequence homology is much higher in those regions thought to be involved in metal binding of the binuclear active site copper of this monooxygenase.
Amino Acid Sequence↗
(Cu,Zn)-metallothioneins from fetal bovine liver. Chemical and spectroscopic properties.
Two metallothioneins (MTs) from bovine fetal liver were purified by a combination of gel filtration and ion-exchange chromatography. The primary structures of the isoproteins MT-1 and MT-2 were elucidated by peptide and amino acid sequence analysis. The amino-terminal part was deduced from automated Edman degradations of the pyridylethylated CNBr-cleaved derivatives. The remaining part of the sequence was established by a comparison of the carboxamidomethylated tryptic peptides to those from equine liver MT-1A and MT-2B. Peptides differing in either amino acid composition or retention time from high pressure liquid chromatography were further subjected to manual Edman degradations or carboxypeptidase Y digestion. The two isoproteins consist of 61 amino acids and show a sequence identity of 90%. When compared with the primary structures of other mammalian MTs, the 20 cysteinyl residues are totally conserved, in agreement with their function as metal ligands. The two isoproteins contain Cu and Zn at a ratio of 3:4. Spectroscopic data reveal absorption properties typical for both Cu- and Zn-thiolate transitions. The marked differences of MT-1 and MT-2 in the Cu-thiolate CD features can be attributed to the six amino acid substitutions occurring exclusively in the amino-terminal parts of the molecules. It is proposed that in bovine fetal MTs also the three copper ions are preferentially bound to the first 9 cysteinyl residues (cluster B) and the four zinc ions to the remaining 11 cysteinyl residues (cluster A) suggested previously by 113Cd NMR spectroscopy of calf liver MTs (Briggs, R. W., and Armitage, I. M. (1982) J. Biol. Chem. 257, 1259-1262).
Amino Acid Sequence↗
Primary structure of copper-zinc superoxide dismutase from Neurospora crassa.
The complete amino acid sequence of copper-zinc superoxide dismutase from Neurospora crassa is reported. The subunit consists of 153 amino acids and has a Mr of 15,850. The primary structure was determined by automated and manual sequence analysis of peptides obtained by digestions of the carboxymethylated and aminoethylated enzyme with trypsin and thermolysin. The protein is devoid of tryptophan and methionine and displays a free amino terminus. Comparison of the amino acid sequence with those from human erythrocyte, bovine erythrocyte, horse liver, swordfish liver, and yeast copper-zinc superoxide dismutases reveals a high degree of sequence homology among the six enzymes. Most prominently, the regions containing the amino acid residues participating in the metal-binding and the half-cystine residues forming the intramolecular disulfide bridge are highly conserved. The invariant amino acids Pro 74 and Asp 76 of the four vertebrate and yeast superoxide dismutases were found to be substituted by arginine and alanine, respectively, in the Neurospora enzyme. These radical substitutions occurring in the zinc ligand region, known to form a characteristic loop structure in bovine erythrocyte copper-zinc superoxide dismutase (Tainer, J. A., Getzoff, E. D., Beem, K. M., Richardson, J. S., and Richardson, D. C. (1982) J. Mol. Biol. 160, 181-217), however, do not affect the catalytic properties of the Neurospora enzyme.
Amino Acid Sequence↗
Isolation and structural organization of the Neurospora crassa copper metallothionein gene.
The Neurospora crassa copper metallothionein gene was cloned and its complete nucleotide sequence is reported. Enriched metallothionein mRNA was used as a template for cDNA synthesis, primed by a metallothionein-specific, synthetic undecanucleotide. The sequence of the cDNA obtained allowed the synthesis of a unique 21-mer which was used to screen a genomic DNA library of N. crassa. In agreement with the published amino acid sequence, the gene codes for a polypeptide 26 amino acid residues in length. The coding region is interrupted by a small intron (94 nucleotides). The gene structure is compared with those of mammalian metallothioneins. In both cases, the coding regions are split by introns, the intron-exon boundaries, however, are in different positions. The neurospora copper metallothionein gene is, to our knowledge, the smallest gene interrupted by an intron isolated so far.
Amino Acid Sequence↗