Search Results (5)

Tyrosinase serves as the key enzyme in melanin biosynthesis, catalyzing the initial steps of the pathway, the hydroxylation of the amino acid L-tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA), followed by the subsequent oxidation of L-DOPA into dopaquinone (DQ), and it facilitates the conversion of 5,6-dihydroxyindole-2-carboxylic acid (DHICA) into 5,6-indolequinone-2-carboxylic acid (IQCA) and 5,6-dihydroxy indole (DHI) into indolequinone (IQ). Despite its versatile substrate capabilities, the precise mechanism underlying tyrosinase’s multi-substrate activity remains unclear. Previously, we expressed, purified, and characterized the recombinant intra-melanosomal domain of human tyrosinase (rTyr). Here, we demonstrate that rTyr mimics native human tyrosinase’s catalytic activities in vitro and in silico. Molecular docking and molecular dynamics (MD) simulations, based on rTyr’s homology model, reveal variable durability and binding preferences among tyrosinase substrates and products. Analysis of root mean square deviation (RMSD) highlights the significance of conserved residues (E203, K334, F347, and V377), which exhibit flexibility during the ligands’ binding. Additionally, in silico analysis demonstrated that the OCA1B-related P406L mutation in tyrosinase substantially influences substrate binding, as evidenced by the decreased number of stable ligand conformations. This correlation underscores the mutation’s impact on substrate docking, which aligns with the observed reduction in rTyr activity. Our study highlights how rTyr dynamically adjusts its structure to accommodate diverse substrates and suggests a way to modulate rTyr ligand plasticity. Full article

The inherited disorder oculocutaneous albinism type 1 (OCA1) is caused by mutations in the TYR gene encoding tyrosinase (Tyr), an enzyme essential to producing pigments throughout the human body. The intramelanosomal domain of Tyr consists of the cysteine-rich and tyrosinase catalytic subdomains, which are essential for enzymatic activity. In protein unfolding, the roles of these subdomains are not well established. Here, we performed six molecular dynamics simulations at room temperature for Tyr and OCA1-related mutant variants P406L and R402Q intramelanosomal domains. The proteins were simulated for 1 μs in water and urea to induce unfolding. In urea, we observed increases in surface area, decreases in intramolecular hydrogen bonding, and decreases in hydrophobic interactions, suggesting a ‘molten globule’ state for each protein. Between all conditions, the cysteine-rich subdomain remains stable, whereas the catalytic subdomain shows increased flexibility. This flexibility is intensified by the P406L mutation, while R402Q increases the catalytic domain’s rigidity. The cysteine-rich subdomain is rigid, preventing the protein from unfolding, whereas the flexibility of the catalytic subdomain accommodates mutational changes that could inhibit activity. These findings match the conclusions from our experimental work suggesting the function alteration by the P406L mutation, and the potential role of R402Q as a polymorphism. Full article

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 °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. Full article

Tyrosinase-related protein 2 (Tyrp2) is involved in the melanogenesis pathway, catalyzing the tautomerization of dopachrome to 5,6-dihydroxyindole-2-carboxylic acid (DHICA). Recently, a new type of albinism was discovered with disease-causing mutations in the TYRP2 gene. Here, for the first time, we characterized the intra-melanosomal protein domain of Tyrp2 (residues 1-474) and missense variants C40S and C61W, which mimic the alterations found in genetic studies. Recombinant proteins were produced in the Trichoplusia Ni (Ti. Ni) larvae, purified by a combination of immobilized metal affinity (IMAC) and gel-filtration (GF) chromatography, and biochemically characterized. The mutants showed the protein expression in the lysates such as the wild type; however, undetectable protein yield after two steps of purification exhibited their misfolding and instability. In addition, the misfolding effect of the mutations was confirmed computationally using homology modeling and molecular docking. Together, experiments in vitro and computer simulations indicated the critical role of the Cys-rich domain in the Tyrp2 protein stability. The results are consistent with molecular modeling, global computational mutagenesis, and clinical data, proving the significance of genetic alterations in cysteine residues, which could cause oculocutaneous albinism type 8. Full article

Pigmentation is the result of a complex process by which the biopolymer melanin is synthesized and packed into melanosomes of melanocytes. Various types of oculocutaneous albinism (OCA), a series of autosomal recessive disorders, are associated with reduced pigmentation in the skin, eyes, and hair due to genetic mutations of proteins involved in melanogenesis. Human tyrosinase (Tyr) and tyrosinase-related protein 1 (Tyrp1) drives the enzymatic process of pigment bio-polymerization. However, within the melanogenic pathway, Tyrp1 has catalytic functions not clearly defined and distinct from Tyr. Here, we characterize the biochemical and biophysical properties of recombinant human Tyrp1. For this purpose, we purified and analyzed the intra-melanosomal domain (Tyrp1tr) for protein stability and enzymatic function in conditions mimicking the environment within melanosomes and the endoplasmic reticulum. The study suggests that Tyrp1tr is a monomeric molecule at ambient temperatures and below (<25 °C). At higher temperatures, >31 °C, higher protein aggregates form with a concurrent decrease of monomers in solution. Also, Tyrp1tr diphenol oxidase activity at pH 5.5 rises as both the pre-incubation temperature and the higher molecular weight protein aggregates formation increases. The enhanced protein activity is consistent with the volume exclusion change caused by protein aggregates. Full article