Coding

Part:BBa_K4414006

Designed by: Yue Yin   Group: iGEM22_NUDT_CHINA   (2022-09-22)

TYR


Usage and Biology

Usage and Biology Tyrosinase(TYR) is a key enzyme in melanin synthesis that catalyzes the rate-limiting step of the reaction. By activating tyrosine kinase can increase the yield of melanin, forming melanin deposition . The melanogenesis process is initiated with the oxidation of L-tyrosine to dopaquinone (DQ) by TYR. The resulting quinone will serve as a substrate for the synthesis of eumelanin and pheomelanin(Hearing & Jiménez, 1987). The formation of DQ is a rate-limiting step in the melanin synthesis because remainder of the reaction sequence can proceed spontaneously at a physiological pH value(Halaban et al., 2002). After DQ formation, it undergoes intramolecular cyclization to produce indoline, leukodopachrome (cyclodopa). The redox exchange between leukodopachrome and DQ give rise to dopachrome and L-3,4-dihydroxyphenylalanine (L-DOPA), which is also a substrate for TYR and oxidized to DQ again by the enzyme. Dopachrome gradually decomposes to give dihydroxyindole (DHI) and dihydroxyindole-2-carboxylicacid (DHICA). The later process is catalyzed by TRP-2, now known as dopachrome tautomerase (DCT). Ultimately, these dihydroxyindoles (DHI and DHICA) are oxidized to eumelanin. TRP-1 is believed to catalyze the oxidation of DHICA to eumelanin. Alongside, DQ is converted to 5-S-cysteinyldopa or glutothionyldopa in the presence of cysteine or glutathione. Subsequent oxidation gives benzothiazine intermediates and finally to produce pheomelanin. Although three enzymes, TYR, TRP-1 and TRP-2 are involved in the melanogenesis pathway, tyrosinase is exclusively necessary for melanogenesis(Pillaiyar et al., 2017). We obtained this DNA by PCR amplification from human cDNA.This part can be used in level 1 biological laboratory.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Fuctional Validation and result

We introduce the plasmid with TYR into the cells, where melanogen is produced as shown.


Figure 1: (Left)The blackening of HEK-293T cells at 24 h or 48 h post glucocorticoids treatment in microscope.(Right)The blackening of HEK-293T cells at 24 h or 48 h post glucocorticoids treatment under the naked eye.

Reference

1. Halaban, R., Patton, R. S., Cheng, E., Svedine, S., Trombetta, E. S., Wahl, M. L., Ariyan, S., & Hebert, D. N. (2002). Abnormal acidification of melanoma cells induces tyrosinase retention in the early secretory pathway. The Journal of Biological Chemistry, 277(17), 14821–14828. https://doi.org/10.1074/jbc.M111497200

2. Hearing, V. J., & Jiménez, M. (1987). Mammalian tyrosinase—The critical regulatory control point in melanocyte pigmentation. The International Journal of Biochemistry, 19(12), 1141–1147. https://doi.org/10.1016/0020-711x(87)90095-4

3. Pillaiyar, T., Manickam, M., & Namasivayam, V. (2017). Skin whitening agents: medicinal chemistry perspective of tyrosinase inhibitors. Journal of Enzyme Inhibition and Medicinal Chemistry, 32, 403 - 425.

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