PMID- 36614088
OWN - NLM
STAT- MEDLINE
DCOM- 20230110
LR  - 20230111
IS  - 1422-0067 (Electronic)
IS  - 1422-0067 (Linking)
VI  - 24
IP  - 1
DP  - 2022 Dec 30
TI  - In Vitro Reconstitution of the Melanin Pathway's Catalytic Activities Using 
      Tyrosinase Nanoparticles.
LID - 10.3390/ijms24010639 [doi]
LID - 639
AB  - 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-Menten 
      kinetics, the diphenol oxidase activity of tyrosinase achieved the maximum 
      production of native dopachrome at 10 min of incubation at 37  degrees 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.
FAU - Osuna, Isabella
AU  - Osuna I
AD  - National Eye Institute, National Institutes of Health, Bethesda, MD 20891, USA.
FAU - Dolinska, Monika B
AU  - Dolinska MB
AD  - National Eye Institute, National Institutes of Health, Bethesda, MD 20891, USA.
FAU - Sergeev, Yuri V
AU  - Sergeev YV
AUID- ORCID: 0000-0002-7204-6572
AD  - National Eye Institute, National Institutes of Health, Bethesda, MD 20891, USA.
LA  - eng
GR  - , ZIA EY000476-10/EY/NEI NIH HHS/United States
PT  - Journal Article
DEP - 20221230
PL  - Switzerland
TA  - Int J Mol Sci
JT  - International journal of molecular sciences
JID - 101092791
RN  - 0 (Melanins)
RN  - EC 1.14.18.1 (Monophenol Monooxygenase)
RN  - 3571-34-4 (dopachrome)
RN  - 0 (Indolequinones)
SB  - IM
MH  - Humans
MH  - *Melanins/metabolism
MH  - Monophenol Monooxygenase/metabolism
MH  - *Indolequinones
MH  - Oxidation-Reduction
PMC - PMC9820814
OTO - NOTNLM
OT  - intra-melanosomal domains of tyrosinases
OT  - melanogenic pathway
OT  - native dopachrome
OT  - oculocutaneous albinism
OT  - tyrosinase domain immobilized to magnetic beads
COIS- The authors declare no conflict of interest. The funders had no role in the 
      design of the study; in the collection, analyses, or interpretation of data; in 
      the writing of the manuscript; or in the decision to publish the results.
EDAT- 2023/01/09 06:00
MHDA- 2023/01/11 06:00
PMCR- 2022/12/30
CRDT- 2023/01/08 01:24
PHST- 2022/11/25 00:00 [received]
PHST- 2022/12/24 00:00 [revised]
PHST- 2022/12/26 00:00 [accepted]
PHST- 2023/01/08 01:24 [entrez]
PHST- 2023/01/09 06:00 [pubmed]
PHST- 2023/01/11 06:00 [medline]
PHST- 2022/12/30 00:00 [pmc-release]
AID - ijms24010639 [pii]
AID - ijms-24-00639 [pii]
AID - 10.3390/ijms24010639 [doi]
PST - epublish
SO  - Int J Mol Sci. 2022 Dec 30;24(1):639. doi: 10.3390/ijms24010639.