| Project/Area Number |
09672205
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Physical pharmacy
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| Research Institution | Fukuyama University |
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Principal Investigator |
HIROSE Junzo Faculty of Engineering, Fukuyama University, Professor, 工学部, 教授 (70080215)
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| Co-Investigator(Kenkyū-buntansha) |
IWAMOTO Hiroyuki Faculty of Engineering, Fukuyama University, Assistant Professor, 工学部, 講師 (90213321)
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| Project Period (FY) |
1997 – 1999
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| Project Status |
Completed (Fiscal Year 1999)
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| Budget Amount *help |
¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1999: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1998: ¥500,000 (Direct Cost: ¥500,000)
Fiscal Year 1997: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Keywords | Bilirubin Oxidase / Multi-copper Enzyme / Copper Enzyme / Copper Protein / ビリルビンオキシダーゼ / アスコルビン酸オキシダーゼ / ビリルビン・オキシダーゼ / 遺伝子工学 / 発現 / 基質特異性 |
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| Research Abstract |
We isolated the clone of bilirubin oxidase (BOD) obtained from Trachyderma Tsunodae K-2593 and deduced the amino acid sequence. The sequence has submitted to the DDDBJ/EMBL/GenBank database as AB 006824. On the basis of EPR, visible spectra, and the copper content of BOD, it was shown that of BOD is multi-copper enzyme, which has one type 2 copper, one type 1 copper, and two type 3 copper. The coordination residues of type 1, type2 and type 3 copper was predicted of homology of the amino acid sequence between BOD and ascorbate oxidase obtained from zucchini. Every ligand of type 2 and type 3 copper ions in BOD was shown to be His and was completely consistent with those of zucchini ascorbate oxidase. The ligand of type 1 copper in BOD was Cys, His, His, and Phe. Met which coordinates with type 1 copper in zucchini ascorbate oxidase was replaced by Phe in BOD. The redox potential of type 1 copper of BOD was measured under anaerobic conditions and was 642 mV that was much higher than that zucchini ascorbate oxidase. The higher redox potential of type 1 copper is because the hydrophobicity of Phe around type 1 copper in BOD raises the redox potential. The various anions influenced the visible spectra of type 1 copper in BOD. NィイD23ィエD2ィイD1-ィエD1 and SCNィイD1-ィエD1 gave the increase of the visible absorption spectra, but FィイD1-ィエD1 gave the decrease of it. EPR spectra of the adducts of these anions with BOD clearly indicated that these anion bind to type 2 copper in BOD. These behaviors were interpreted by the disproportination of the electron between type 1 copper and type 2/type 3 cluster. We tried the expression of the gene of BOD in Ecoli, but it was not succeeded.
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