Molecular Modeling of the Multiple-Substrate Activity of the Human Recombinant Intra-Melanosomal Domain of Tyrosinase and Its OCA1B-Related Mutant Variant P406L.

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.