title: A three-dimensional model of mammalian tyrosinase active site accounting for loss of function mutations.
abstract: Tyrosinases are the first and rate-limiting enzymes in the synthesis of melanin pigments responsible for colouring hair, skin and eyes. Mutation of tyrosinases often decreases melanin production resulting in albinism, but the effects are not always understood at the molecular level. Homology modelling of mouse tyrosinase based on recently published crystal structures of non-mammalian tyrosinases provides an active site model accounting for loss-of-function mutations. According to the model, the copper-binding histidines are located in a helix bundle comprising four densely packed helices. A loop containing residues M374, S375 and V377 connects the CuA and CuB centres, with the peptide oxygens of M374 and V377 serving as hydrogen acceptors for the NH-groups of the imidazole rings of the copper-binding His367 and His180. Therefore, this loop is essential for the stability of the active site architecture. A double substitution (374)MS(375) --> (374)GG(375) or a single M374G mutation lead to a local perturbation of the protein matrix at the active site affecting the orientation of the H367 side chain, that may be unable to bind CuB reliably, resulting in loss of activity. The model also accounts for loss of function in two naturally occurring albino mutations, S380P and V393F. The hydroxyl group in S380 contributes to the correct orientation of M374, and the substitution of V393 for a bulkier phenylalanine sterically impedes correct side chain packing at the active site. Therefore, our model explains the mechanistic necessity for conservation of not only active site histidines but also adjacent amino acids in tyrosinase.
authors.affiliation: Institute of Molecular Biophysics, University of Mainz, Mainz, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Schweikardt
authors.forename: Concepción
authors.initials: C
authors.lastname: Olivares
authors.forename: Francisco
authors.initials: F
authors.lastname: Solano
authors.forename: Elmar
authors.initials: E
authors.lastname: Jaenicke
authors.forename: José Carlos
authors.initials: JC
authors.lastname: García-Borrón
authors.forename: Heinz
authors.initials: H
authors.lastname: Decker
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001671:Biochemistry / Q000379:methods; D006639:Histidine / Q000737:chemistry; D006801:Humans; D008543:Melanins / Q000737:chemistry; D051379:Mice; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010649:Phenylalanine / Q000737:chemistry; D013302:Streptomyces / Q000378:metabolism
chemical_list: D008543:Melanins; D010649:Phenylalanine; D006639:Histidine; D014442:Monophenol Monooxygenase