In Vitro Reconstitution of the Melanin Pathway’s Catalytic Activities Using Tyrosinase Nanoparticles
The melanogenesis pathway is characterized by a series of reactions catalyzed by key enzymes, such as tyrosinase (TYR), tyrosinase-related protein 2 (TYRP2), and tyrosinase-related protein 1 (TYRP1), to produce melanin pigment. However, in vitro studies of the catalytic activity were incomplete beca...
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| Format: | Article |
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MDPI AG
2022-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/1/639 |
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| author | Isabella Osuna Monika B. Dolinska Yuri V. Sergeev |
| author_facet | Isabella Osuna Monika B. Dolinska Yuri V. Sergeev |
| author_sort | Isabella Osuna |
| collection | DOAJ |
| description | The melanogenesis pathway is characterized by a series of reactions catalyzed by key enzymes, such as tyrosinase (TYR), tyrosinase-related protein 2 (TYRP2), and tyrosinase-related protein 1 (TYRP1), to produce melanin pigment. However, in vitro studies of the catalytic activity were incomplete because of a lack of commercially available enzyme substrates, such as dopachrome. Herein, human recombinant intra-melanosomal domains of key enzymes were produced in <i>Trichoplusia ni (T. ni)</i> larvae and then purified using a combination of chromatography techniques in catalytically active form. Using Michaelis–Menten kinetics, the diphenol oxidase activity of tyrosinase achieved the maximum production of native dopachrome at 10 min of incubation at 37 °C for TYR immobilized to magnetic beads (TYR-MB). The presence of dopachrome was confirmed spectrophotometrically at 475 nm through HPLC analysis and in the TYRP2-catalyzed reaction, yielding 5,6-dihydroxyindole-2-carboxylic acid (DHICA). In the TYRP1-driven oxidation of DHICA, the formation of 5,6-indolequinone-2-carboxylic acid (IQCA) was confirmed at ~560 nm. This is the first in vitro reconstitution of the reactions from the melanogenic pathway based on intra-melanosomal domains. In the future, this approach could be used for quantitative in vitro analysis of the melanin pathway, biochemical effects associated with inherited disease-related mutations, and drug screens. |
| first_indexed | 2024-03-11T09:58:59Z |
| format | Article |
| id | doaj.art-7e77f3a14a114a20aadbd727ee20d058 |
| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-11T09:58:59Z |
| publishDate | 2022-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | International Journal of Molecular Sciences |
| spelling | doaj.art-7e77f3a14a114a20aadbd727ee20d0582023-11-16T15:36:23ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672022-12-0124163910.3390/ijms24010639In Vitro Reconstitution of the Melanin Pathway’s Catalytic Activities Using Tyrosinase NanoparticlesIsabella Osuna0Monika B. Dolinska1Yuri V. Sergeev2National Eye Institute, National Institutes of Health, Bethesda, MD 20891, USANational Eye Institute, National Institutes of Health, Bethesda, MD 20891, USANational Eye Institute, National Institutes of Health, Bethesda, MD 20891, USAThe melanogenesis pathway is characterized by a series of reactions catalyzed by key enzymes, such as tyrosinase (TYR), tyrosinase-related protein 2 (TYRP2), and tyrosinase-related protein 1 (TYRP1), to produce melanin pigment. However, in vitro studies of the catalytic activity were incomplete because of a lack of commercially available enzyme substrates, such as dopachrome. Herein, human recombinant intra-melanosomal domains of key enzymes were produced in <i>Trichoplusia ni (T. ni)</i> larvae and then purified using a combination of chromatography techniques in catalytically active form. Using Michaelis–Menten kinetics, the diphenol oxidase activity of tyrosinase achieved the maximum production of native dopachrome at 10 min of incubation at 37 °C for TYR immobilized to magnetic beads (TYR-MB). The presence of dopachrome was confirmed spectrophotometrically at 475 nm through HPLC analysis and in the TYRP2-catalyzed reaction, yielding 5,6-dihydroxyindole-2-carboxylic acid (DHICA). In the TYRP1-driven oxidation of DHICA, the formation of 5,6-indolequinone-2-carboxylic acid (IQCA) was confirmed at ~560 nm. This is the first in vitro reconstitution of the reactions from the melanogenic pathway based on intra-melanosomal domains. In the future, this approach could be used for quantitative in vitro analysis of the melanin pathway, biochemical effects associated with inherited disease-related mutations, and drug screens.https://www.mdpi.com/1422-0067/24/1/639melanogenic pathwayoculocutaneous albinismintra-melanosomal domains of tyrosinasestyrosinase domain immobilized to magnetic beadsnative dopachrome |
| spellingShingle | Isabella Osuna Monika B. Dolinska Yuri V. Sergeev In Vitro Reconstitution of the Melanin Pathway’s Catalytic Activities Using Tyrosinase Nanoparticles International Journal of Molecular Sciences melanogenic pathway oculocutaneous albinism intra-melanosomal domains of tyrosinases tyrosinase domain immobilized to magnetic beads native dopachrome |
| title | In Vitro Reconstitution of the Melanin Pathway’s Catalytic Activities Using Tyrosinase Nanoparticles |
| title_full | In Vitro Reconstitution of the Melanin Pathway’s Catalytic Activities Using Tyrosinase Nanoparticles |
| title_fullStr | In Vitro Reconstitution of the Melanin Pathway’s Catalytic Activities Using Tyrosinase Nanoparticles |
| title_full_unstemmed | In Vitro Reconstitution of the Melanin Pathway’s Catalytic Activities Using Tyrosinase Nanoparticles |
| title_short | In Vitro Reconstitution of the Melanin Pathway’s Catalytic Activities Using Tyrosinase Nanoparticles |
| title_sort | in vitro reconstitution of the melanin pathway s catalytic activities using tyrosinase nanoparticles |
| topic | melanogenic pathway oculocutaneous albinism intra-melanosomal domains of tyrosinases tyrosinase domain immobilized to magnetic beads native dopachrome |
| url | https://www.mdpi.com/1422-0067/24/1/639 |
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