J. Biochem, 2003, Vol. 133, No. 6 767-772
© 2003 Japanese Biochemical Society
BIOCHEMISTRY |
Type III Cu Mutants of Myrothecium verrucaria Bilirubin Oxidase
1 Department of Chemistry, Faculty of Science and Technology, Aoyama Gakuin University, Chitosedai, Setagaya, Tokyo 157-8572; 2 Kyoto Pharmaceutical University, 5 Nakauti-cho, Misasagi, Yamashina-ku, Kyoto 607-8414; 3 Development Division, Amano Enzyme Co., Ltd., Nishiharu, Nishi-kasugai, Aichi 481-0041, 4 Institute of Natural Sciences, Nagoya City University, Yamanohata 1, Mizuho, Nagoya 467-8501; and 5 Department of Chemistry, Faculty of Science, Kanazawa University, Kakuma, Kanazawa 920-1192
Type III Cu ligand, His456 and His458, of Myrothecium verrucaria (MT-1) bilirubin oxidases (BO) [EC 1.3.3.5] were doubly mutated as to Lys, Asp, and Val. In spite of perturbation of the type III Cu centers, these mutants were pale blue or colourless when isolated. However, they became intense blue on reaction with reducing agents such as dithionite, ascorbate, hexacyanoferrate(II), and octacyanotangstate(IV) under air, or with an oxidizing agent such as hexacyanoferrate(III), indicating that they are in mixed forms when expressed in Aspergillus oryzae. His456.458Lys and His456.458Asp mutated as to potential coordinating groups showed weak BO and ferroxidase activities, while His 456.458Val mutated as to non-coordinating groups showed no enzyme activity at all.
+ Present address: Department of Molecular Biology, Keio University School of Medicine, 35, Shinanomachi, Shinjuku-ku, Tokyo, 160-8582.
To whom correspondence should be addressed. Tel: +81-76-264-5685, Fax: +81-264-5742, E-mail: ts0513{at}kenroku.kanazawa-u.ac.jp