Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis

Copper ligands of the recombinant tyrosinase from the fungus Aspergillus or yzae expressed in Saccharomyces cerevisiae or Escherichia coli were identif ied by site-directed mutagenesis. The recombinant protyrosinases expressed in S. cerevisiae were assayed for catalytic activities of mono-oxygenase an d L-dopa oxidase at pH 5.5 after acid shock at pH 3.0. Replacements of His- 63, His-84, His-93, His-290, His-394, His-332 or His-333 with asparagine re sulted in mutant enzymes exhibiting no activities. The site-directed mutant Cys82Ala showed that Cys-82 was also an essential residue for the activity . We obtained homogeneous preparations of activated tyrosinases From mutate d thioredoxin fusion gene products expressed in E. coli by acid shock. The copper contents of engineered mutants and wild-type enzyme expressed in E. coli were determined by atomic absorption spectrophotometry. The wild-type enzyme contained 2 g-atoms of copper/mol of the subunit. The His63Asn, His8 4Asn, His93Asn, His290Asn, His294Asn, His332Asn, His333Asn or Cys82Ala subs titution decreased copper binding by approx. 50%, indicating that the mutan ts contain only approx. 1 g-atom of copper/mol of the subunit. The five mut ants His63Asn, His93Asn, His290Asn, His294Asn and Cys82Ala contain only one copper ion, which is fully detectable by EPR. From the correlation of g(pa rallel to) and (Cu)A(parallel to), we deduced that the nitrogen or sulphur donors in the copper ligands should be in a square or a distorted tetrahedr al geometric environment. In further atomic absorption spectrophotometry ex periments, no copper atom was observed in the seven double mutants His63Asn /His290Asn, His63Asn/His294Asn, His63Asn/His332Asn, His63Asn/His333Asn, wCy s82Ala/His290Asn, His84Asn/His333Asn and His93Asn/His290Asn. We propose a n ew structure of active sites of tyrosinase from A. oryzae: the most likely binding sites of tyrosinase for Cu(A) are His-63, His-84 and His-93, with t he remaining conserved Cys-82 providing the fourth ligand. Cu(B) liganded b y four histidine residues, His-290, His-294, His-332 and His-333, is identi fied as new binding motif of Cu(B).