Papers by francisco solano
Pigm Cell Res, 2003
ABSTRACT Tyrosinase (Tyr), the rate-limiting melanogenic enzyme, is a copper-containing glycoprot... more ABSTRACT Tyrosinase (Tyr), the rate-limiting melanogenic enzyme, is a copper-containing glycoprotein with a complex post-translational processing that involves N-glycosylation in several sites, including one located in the CuB metal binding site, exit from the endoplasmic reticulum to the Golgi, copper binding and sorting to the melanosome. Aberrant processing is causally related to depigmented phenotypes. Two other highly similar proteins, Tyrp1 and Dct, also contribute to normal melanogenesis. These proteins share many structural features, including six potential glycosylation sequons, and two highly conserved metal binding sites, that bind Zinc in Dct and a still unidentified metal ion in Tyrp1. In order to identify structural elements and specific interactions responsible for metal ion specificity and proper processing, we constructed several chimeric genes derived from specific domains of mouse Tyr and Tyrp1. After transient expression, we analyzed the enzymatic activity and glycosylation pattern of the resulting proteins, as compared Tyr in a natural melanocytic environment. Replacement of the metal binding site B (MeB) of Tyr by that of Tyrp1 led to an enzymatically inactive and missfolded chimeric protein (CstB). Glycosylation sequon occupancy was analysed by partial digestion with PNGase F and electrophoresis. Tyr was glycosylated in all six potential sites, whereas two glycosylation sequons were unoccupied in CstB. Unexpectedly, the MeB site was efficiently glycosylated in CstB. The data are consistent with the presence of two conformation-dependent glycosylation sites in Tyr, related to an essential interaction involving MeB. Attempts to rescue proper folding and enzyme activity by point mutation of Tyrp1-specific residues in MeB back to the Tyr-specific amino acid were unsuccessful, probably implying that this interaction is multipoint and involves several side chains. A Tyr-derived chimera where the CuA site of Tyr was replaced by the MeA of Tyrp1 retained some enzymatic activity (25% tyrosine hydroxylase and 10% DOPA oxidase) and was efficiently glycosylated. This shows that the MeA site of Tyrp1 is able to bind copper and sustain the typical Tyr enzymatic activities, supporting that Typ1 might be a low specific activity Tyr isoenzyme. Acknowledgements: Supported by grants PM99-0138 and BIO2001-0140
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Biochimica Et Biophysica Acta Protein Structure and Molecular Enzymology, May 5, 2001
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Biochemical Pharmacology, Feb 1, 1988
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Journal of Cell Science, Jun 15, 2001
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Bba Proteins Proteomics, 2006
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Microbiology Sgm, 2003
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Biochimica Et Biophysica Acta Protein Structure and Molecular Enzymology, Jan 11, 1994
Dopachrome tautomerase (DCT) is a recently characterized enzyme contributing to the control of me... more Dopachrome tautomerase (DCT) is a recently characterized enzyme contributing to the control of melanogenesis in mammals. The enzyme catalyzes the rearrangement of L-Dopachrome (L-DC) to 5,6-dihydroxyindole 2-carboxylic acid (DHICA), while the spontaneous rearrangement of L-DC leads to 5,6-dihydroxyindole (DHI). Due to the lower reactivity of DHICA in comparison to DHI, DCT could provide a protective mechanism against the cytotoxicity of decarboxylated indolic melanogenic intermediates by limiting the formation of these highly reactive decarboxylated species within melanocytes. We have followed the binding of radioactive melanogenic precursors to a model protein, bovine serum albumin (BSA). Using L-DC as initial melanin precursor, this binding was decreased by DCT in a concentration-dependent manner. In the presence of tyrosinase, the binding of L-Dopa-derived intermediates to BSA was also decreased by DCT and the percentage of decrease was even higher than using L-DC as initial melanin precursor. SDS-PAGE followed by fluorographic detection of radioactive bands showed the formation of covalent adducts between BSA and melanin precursors, as well as of aggregated forms of this protein. This aggregation was also diminished by DCT. These data indicate that DCT could play a protective role against the cytotoxic action of decarboxylated indoles within mammalian melanocytes.
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Pigm Cell Res, 2003
ABSTRACT Melanin is the most widely distributed pigment on nature. In higher animals, the main ro... more ABSTRACT Melanin is the most widely distributed pigment on nature. In higher animals, the main roles of this pigment are associated to cutaneous photoprotection and proper function of the eye and inner ear, but in microorganisms melanin seems to be a polymer to enhance the survival and competitive abilities of species in certain environments, and for instance to increase the virulence and pathogenesis to several strains (1). We have studied the correlation between pigment formation and culture conditions, as well as the main enzymatic properties of the polyphenol oxidase system (PPO) in two strains of Ralstonia solanacearum, a devastating plant pathogen with a wide host range. The strains studied were obtained from France (GMI1000, whose genome sequence has been recently completed revealing several putative PPO genes, 2), and Spain (strain CECT125). Both strains have shown different characteristics concerning their PPO systems, although their pigmentation in different media is quite similar. Basically, strain GMI1000 seems to show a multipotent cytosolic PPO acting at acidic optimal pH (around 5). The dopa oxidase (DO) and laccase activities (DMPO) are higher than the tyrosine hydroxylase (TH). The second one seems to show two different PPOs with neutral optimal pH. The laccase activity is mostly membrane-bound, but tyrosinase is cytosolic and it shows a TH activity higher than DO, an interesting difference to all other known tyrosinases. Other comparative studies concerning SDS activation, stability against heat, proteases and sensitivity to PPO inhibitors will also presented. The usefulness of these enzymes as models to approach the requirements of the tyrosinase active site and their putative relationship with pigment formation and pathogenesis would be considered. Acknowledgements: We are grateful to grant BIO2001/1140 to carry out this study.
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Journal of Bacteriology, Jul 1, 2000
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Clinical Chemistry and Enzymology Communications, 1993
RefDoc Refdoc est un service / is powered by. ...
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Mamm Genome, 1999
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J Invest Dermatol, 1992
α-MSH was found to decrease the recently characterized dopachrome tautomerase activity in culture... more α-MSH was found to decrease the recently characterized dopachrome tautomerase activity in cultures of B16/F10 mouse melanoma cells. Other stimulating agents of melanogenesis, like dibutyryl cyclic AMP, 3-isobutyl-1-methylxanthinem theophylline, retinol, and retinoic acid, caused the same effect. The grade of inhibition depended on the nature of the agent and the time of exposure. In all cases, both melanin production and tyrosinase activity were activated by these treatments, although the grade of tyrosine hydroxylase and dopa oxidase stimulation was different. Moreover, no correlation among the intensities of dopachrome tautomerase inhibition and tyrosinase activation by the tested agents could be obtained. The significance of these results in the regulation of mammalian melanogenesis is discussed.
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Biochemical and Biophysical Research Communications, Nov 26, 1997
The recently characterized marine melanogenic bacterium MMB-1 contains a pluripotent polyphenol o... more The recently characterized marine melanogenic bacterium MMB-1 contains a pluripotent polyphenol oxidase (PPO) which catalyzes the oxidation of a very wide range of substrates considered specific for tyrosinase or laccase. This range includes monophenols such as L-tyrosine, o-diphenols such as L-dopa, p-diphenols such as hydroquinone, o-aminophenols such as 3-hydroxyanthranilic acid, activated monophenols such as 2,6-dimethoxyphenol and syringaldazine, and chromophores such as ABTS. This is the first report of an enzyme that is able to catalyze the oxidation of compounds so far considered specific for tyrosinases (L-tyrosine) or laccase (syringaldazine), showing cresolase, catechol oxidase and laccase activities. Such PPO could be a very useful model to study the structural requirements, catalytic mechanisms and involvement of the copper sites existing in non-blue and blue copper-oxidases.
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Pigment Cell Research, Apr 1, 2002
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Applied and Environmental Microbiology, Sep 1, 1997
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Microbiology, Aug 1, 2002
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Biochemistry international
The nature of the essential residues at the active site of Harding-Passey mouse melanoma tyrosina... more The nature of the essential residues at the active site of Harding-Passey mouse melanoma tyrosinase has been explored by kinetic and photochemical modification studies. Km for L-dopa depends strongly on pH, so that acidic pH prevents the formation of the enzyme-substrate complex because the protonation of an enzyme group with a pKa of 6.6. Halide ions inhibit competitively the enzyme activity, being F the more potent one. This inhibition is also pH-dependent, showing the involvement of a protonatable group of the enzyme with apparent pKa ranging from 5.9 to 7.0. Tyrosinase has also been modified with visible light using Rose Bengal as photosensitizer, yielding a pH-dependent photoinactivation, characteristic of histidyl residues. All these results strongly support that histidine plays an important role in the dopa-oxidase activity of the enzyme, very probably acting as the ligand of copper at the active site of the enzyme.
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Journal of Cell Science
ABSTRACT
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The Biochemical journal, Jan 15, 1991
Dopachrome tautomerase (EC 5.3.2.3) catalyses the tautomerization of dopachrome to 5,6-dihydroxyi... more Dopachrome tautomerase (EC 5.3.2.3) catalyses the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA) within the melanin-formation pathway. We have analysed a series of substrate analogues and related compounds as possible substrates and inhibitors of tautomerization. The enzyme appears to be highly specific since D-dopachrome, alpha-methyldopachrome, dopaminochrome, adrenochrome methyl ether and deoxyadrenochrome are not substrates. Conversely, dopachrome tautomerase catalyses the tautomerization of dopachrome methyl ester, suggesting that a carboxy group, either free or as a methyl ester, is essential for enzyme recognition. No inhibition of dopachrome tautomerization was observed in the presence of either semiquinonic compounds, such as tropolone and L-mimosine, or pyrrole-2-carboxylic acid and unsubstituted indole. However, a number of indole derivatives, including DHICA, the product of dopachrome tautomerization, and the analogues 5-hydroxyindole-2-ca...
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Applied and environmental microbiology, 1997
A novel marine melanogenic bacterium, strain MMB-1, was isolated from the Mediterranean Sea. The ... more A novel marine melanogenic bacterium, strain MMB-1, was isolated from the Mediterranean Sea. The taxonomic characterization of this strain indicated that it belongs to the genus Alteromonas. Under in vivo conditions, L-tyrosine was the specific monophenolic precursor for melanin synthesis. This bacterium contained all types of activities associated with polyphenol oxidases (PPOs), cresolase (EC 1.18.14.1), catecholase (EC 1.10.3.1), and laccase (EC 1.10.3.2). These activities were due to the presence of two different PPOs. The first one showed all the enzymatic activities, but it was not involved in melanogenesis in vivo, since amelanogenic mutant strains obtained by nitrosoguanidine treatment contained levels of this PPO similar to that of the wild-type MMB-1 strain. The second PPO showed cresolase and catecholase activities but no laccase, and it was involved in melanogenesis, since this enzyme was lost in amelanogenic mutant strains. This PPO was strongly activated by sodium dode...
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Papers by francisco solano