Multifunctions of MelB, a fungal tyrosinase from Aspergillus oryzae.
ABSTRACT The pro form of melB tyrosinase from the melB gene of Aspergillus oryzae was over-produced from E. coli and formed a homodimer that exhibited the spectral features of met-tyrosinase. In the presence of NH(2)OH (reductant), the proenzyme bound dioxygen to give a stable (μ-η(2):η(2) -peroxo)dicopper(II) species (oxy form), thus indicating that the pro form tyrosinase can function as an oxygen carrier or storage protein like hemocyanin. The pro form tyrosinase itself showed no catalytic activity toward external substrates, but proteolytic digestion with trypsin activated it to induce tyrosinase activity. Mass spectroscopy analyses, mutagenesis experiments, and colorimetry assays have demonstrated that the tryptic digestion induced cleavage of the C-terminal domain (Glu458-Ala616), although the dimeric structure of the enzyme was retained. The structural changes induced by proteolytic digestion might open the entrance to the enzyme active site for substrate incorporation.
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ABSTRACT: Copper active sites play a major role in biological and abiological dioxygen activation. Oxygen intermediates have been studied in detail for the proteins and enzymes involved in reversible O(2) binding (hemocyanin), activation (tyrosinase), and four-electron reduction to water (multicopper oxidases). These oxygen intermediates exhibit unique spectroscopic features indicative of new geometric and electronic structures involved in oxygen activation. The spectroscopic and quantum-mechanical study of these intermediates has defined geometric- and electronic-structure/function correlations, and developed detailed reaction coordinates for the reversible binding of O(2), hydroxylation, and H-atom abstraction from different substrates, and the reductive cleavage of the O-O bond in the formation water.Angewandte Chemie International Edition 01/2002; 40(24):4570-4590. · 13.73 Impact Factor
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ABSTRACT: A new tyrosinase-encoding gene (2,204 bp) and the corresponding cDNA (1,857 nucleotides) from the white-rot fungus Pycnoporus sanguineus BRFM49 were cloned. This gene consisted of seven exons and six introns and encoded a predicted protein of 68 kDa, exceeding the mature tyrosinase by 23 kDa. P. sanguineus tyrosinase cDNA was over-expressed in Aspergillus niger, a particularly suitable fungus for heterologous expression of proteins of biotechnological interest, under the control of the glyceraldehyde-3-phosphate-dehydrogenase promoter as strong and constitutive promoter. The glucoamylase preprosequence of A. niger was used to target the secretion. This construction enabled the production of recombinant tyrosinase in the extracellular medium of A. niger. The identity of the purified recombinant protein was confirmed by N-terminal amino acid sequencing. The maturation process was shown to be effective in A. niger, and the recombinant enzyme was fully active, with a molecular mass of 45 kDa. The best transformant obtained, A. niger D15#26-e, produced extracellular tyrosinase activities of 534 and 1,668 U l(-1) for monophenolase and diphenolase, respectively, which corresponded to a protein yield of ca. 20 mg l(-1).Applied Microbiology and Biotechnology 06/2006; 70(5):580-9. · 3.69 Impact Factor
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ABSTRACT: Catechol oxidases are ubiquitous plant enzymes containing a dinuclear copper center. In the wound-response mechanism of the plant they catalyze the oxidation of a broad range of ortho-diphenols to the corresponding o-quinones coupled with the reduction of oxygen to water. The crystal structures of the enzyme from sweet potato in the resting dicupric Cu(II)-Cu(II) state, the reduced dicuprous Cu(I)-Cu(I) form, and in complex with the inhibitor phenylthiourea were analyzed. The catalytic copper center is accommodated in a central four-helix-bundle located in a hydrophobic pocket close to the surface. Both metal binding sites are composed of three histidine ligands. His 109, ligated to the CuA site, is covalently linked to Cys 92 by an unusual thioether bond. Based on biochemical, spectroscopic and the presented structural data, a catalytical mechanism is proposed in which one of the oxygen atoms of the diphenolic substrate binds to CuB of the oxygenated enzyme.Natural Structural Biology 01/1999; 5(12):1084-90.