title: Albino mutants of Streptomyces glaucescens tyrosinase.
abstract: Site-directed mutagenesis was used to determine the functional role of several residues of Streptomyces glaucescens tyrosinase. Replacement of His-37, -53, -193 or -215 by glutamine yields albino phenotypes, as determined by expression on melanin-indicator plates. The purified mutant proteins display no detectable oxy-enzyme and increased Cu lability at the binuclear active site. The carbonyl derivatives of H189Q and H193Q luminesce, with lambda max. displaced more than 25 nm to a longer wavelength compared with native tyrosinase. The remaining histidine mutants display no detectable luminescence. The results are consistent with these histidine residues (together with His-62 and His-189 reported earlier) acting as Cu ligands in the Streptomyces glaucescens enzyme. Conservative substitution of the invariant Asn-190 by glutamine also gives an albino phenotype, no detectable oxy-enzyme and labilization of active-site Cu. The luminescence spectrum of carbonyl-N190Q, however, closely resembles that of the native enzyme under conditions promoting double Cu occupancy of the catalytic site. A critical role for Asn-190 in active-site hydrogen-bonding interactions is proposed.
authors.affiliation: Biochemisches Institut, Universität Zürich, Switzerland.
authors.forename: M P
authors.initials: MP
authors.lastname: Jackman
authors.forename: A
authors.initials: A
authors.lastname: Hajnal
authors.forename: K
authors.initials: K
authors.lastname: Lerch
mesh_terms: D000417:Albinism / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005998:Glycine / Q000235:genetics; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013302:Streptomyces / Q000201:enzymology*
chemical_list: D006639:Histidine; D014442:Monophenol Monooxygenase; D005998:Glycine