Copper ligands of the recombinant tyrosinase from the fungus Aspergillus oryzae expressed in Saccharomyces cerevisiae or Escherichia coli were identified by site-directed mutagenesis. The recombinant protyrosinases expressed in S. cerevisiae were assayed for catalytic activities of mono-oxygenase and 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-294, His-332 or His-333 with asparagine resulted 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 mutated 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, His84Asn, His93Asn, His290Asn, His294Asn, His332Asn, His333Asn or Cys82Ala substitution decreased copper binding by approx. 50%, indicating that the mutants contain only approx. 1 g-atom of copper/mol of the subunit. The five mutants His63Asn, His93Asn, His290Asn, His294Asn and Cys82Ala contain only one copper ion, which is fully detectable by EPR. From the correlation of g‖ and CuA‖, we deduced that the nitrogen or sulphur donors in the copper ligands should be in a square or a distorted tetrahedral geometric environment. In further atomic absorption spectrophotometry experiments, no copper atom was observed in the seven double mutants His63Asn/His290Asn, His63Asn/His294Asn, His63Asn/His332Asn, His63Asn/His333Asn, Cys82Ala/His290Asn, His84Asn/His333Asn and His93Asn/His290Asn. We propose a new 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 the remaining conserved Cys-82 providing the fourth ligand. Cu(B) liganded by four histidine residues, His-290, His-294, His-332 and His-333, is identified as new binding motif of Cu(B).
Skip Nav Destination
Article navigation
September 2000
-
Cover Image
Cover Image
- PDF Icon PDF LinkTable of Contents
Research Article|
August 23 2000
Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis
Motoyoshi NAKAMURA;
Motoyoshi NAKAMURA
*Laboratory of Molecular Enzymology, Department of Molecular and Cellular Biology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Search for other works by this author on:
Tasuku NAKAJIMA;
Tasuku NAKAJIMA
*Laboratory of Molecular Enzymology, Department of Molecular and Cellular Biology, Division of Life Science, Graduate School of Agricultural Science, Tohoku University, 1-1, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
Search for other works by this author on:
Yasunori OHBA;
Yasunori OHBA
†Institute for Chemical Reaction Science, Tohoku University, Katahira 2-1-1, Sendai 980-8577, Japan
Search for other works by this author on:
Seigo YAMAUCHI;
Seigo YAMAUCHI
†Institute for Chemical Reaction Science, Tohoku University, Katahira 2-1-1, Sendai 980-8577, Japan
Search for other works by this author on:
Byung Rho LEE;
Byung Rho LEE
‡Laboratory of Molecular Enzymology, Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan
Search for other works by this author on:
Eiji ICHISHIMA
Eiji ICHISHIMA
1
‡Laboratory of Molecular Enzymology, Department of Bioengineering, Faculty of Engineering, Soka University, Hachioji, Tokyo 192-8577, Japan
1To whom correspondence should be addressed (e-mail ichisima@t.soka.ac.jp).
Search for other works by this author on:
Publisher: Portland Press Ltd
Received:
December 13 1999
Revision Received:
April 19 2000
Accepted:
June 14 2000
Online ISSN: 1470-8728
Print ISSN: 0264-6021
The Biochemical Society, London © 2000
2000
Biochem J (2000) 350 (2): 537–545.
Article history
Received:
December 13 1999
Revision Received:
April 19 2000
Accepted:
June 14 2000
Citation
Motoyoshi NAKAMURA, Tasuku NAKAJIMA, Yasunori OHBA, Seigo YAMAUCHI, Byung Rho LEE, Eiji ICHISHIMA; Identification of copper ligands in Aspergillus oryzae tyrosinase by site-directed mutagenesis. Biochem J 1 September 2000; 350 (2): 537–545. doi: https://doi.org/10.1042/bj3500537
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.