{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,3]],"date-time":"2026-06-03T14:33:29Z","timestamp":1780497209575,"version":"3.54.1"},"reference-count":27,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2022,12,30]],"date-time":"2022-12-30T00:00:00Z","timestamp":1672358400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"National Eye Institute Intramural Program, NIH","award":["ZIA EY000476-10"],"award-info":[{"award-number":["ZIA EY000476-10"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>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 Trichoplusia ni (T. ni) larvae and then purified using a combination of chromatography techniques in catalytically active form. Using Michaelis\u2013Menten kinetics, the diphenol oxidase activity of tyrosinase achieved the maximum production of native dopachrome at 10 min of incubation at 37 \u00b0C 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.<\/jats:p>","DOI":"10.3390\/ijms24010639","type":"journal-article","created":{"date-parts":[[2022,12,30]],"date-time":"2022-12-30T03:31:17Z","timestamp":1672371077000},"page":"639","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["In Vitro Reconstitution of the Melanin Pathway\u2019s Catalytic Activities Using Tyrosinase Nanoparticles"],"prefix":"10.3390","volume":"24","author":[{"given":"Isabella","family":"Osuna","sequence":"first","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20891, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Monika B.","family":"Dolinska","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20891, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7204-6572","authenticated-orcid":false,"given":"Yuri V.","family":"Sergeev","sequence":"additional","affiliation":[{"name":"National Eye Institute, National Institutes of Health, Bethesda, MD 20891, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"41","DOI":"10.1111\/pcmr.12546","article-title":"Oculocutaneous albinism type 1: Link between mutations, tyrosinase conformational stability, and enzymatic activity","volume":"30","author":"Dolinska","year":"2017","journal-title":"Pigment Cell Melanoma Res."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"479","DOI":"10.1038\/s41436-020-00997-8","article-title":"Dopachrome tautomerase variants in patients with oculocutaneous albinism","volume":"23","author":"Pennamen","year":"2021","journal-title":"Genet. 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