title: Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.
abstract: BACKGROUND
  Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes.
  
  
  RESULTS
  The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure.
  
  
  CONCLUSIONS
  The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.
authors.affiliation: National Eye Institute, NIH, Bethesda, Maryland, United States of America.
authors.forename: Monika B
authors.initials: MB
authors.lastname: Dolinska
authors.affiliation: Chesapeake PERL, Savage, Maryland, United States of America.
authors.forename: Elena
authors.initials: E
authors.lastname: Kovaleva
authors.affiliation: Eunice Kennedy Shriver National Institute Child Health and Human Development, NIH, Bethesda, Maryland, United States of America.
authors.forename: Peter
authors.initials: P
authors.lastname: Backlund
authors.affiliation: National Institute of Arthritis and Musculoskeletal and Skin Diseases, NIH, Bethesda, Maryland, United States of America.
authors.forename: Paul T
authors.initials: PT
authors.lastname: Wingfield
authors.affiliation: National Eye Institute, NIH, Bethesda, Maryland, United States of America.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Brooks
authors.affiliation: National Eye Institute, NIH, Bethesda, Maryland, United States of America.
authors.forename: Yuri V
authors.initials: YV
authors.lastname: Sergeev
mesh_terms: D000417:Albinism / Q000201:enzymology* / Q000235:genetics*; D000595:Amino Acid Sequence; D002384:Catalysis; D002942:Circular Dichroism; D004789:Enzyme Activation; D015870:Gene Expression; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase