| Project/Area Number |
13125202
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| Research Category |
Grant-in-Aid for Scientific Research on Priority Areas
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| Allocation Type | Single-year Grants |
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| Review Section |
Science and Engineering
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| Research Institution | SHIGA UNIVERSITY OF MEDICAL SCIENCE |
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Principal Investigator |
HORIIKE Kihachiro Shiga Univ. of Med. Sci., Department of Biochemistry, Professor, 医学部, 教授 (80089870)
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| Co-Investigator(Kenkyū-buntansha) |
KIMURA Takahide KIMURA,Takahide, 医学部, 教授 (70167378)
TANAKA Hiroyuki TANAKA,Hiroyuki, 医学部, 助手 (10293820)
ISHIDA Tetsuo ISHIDA,Tetsuo, 医学部, 助教授 (10176191)
KITA Akiko Kyoto Univ., Graduate School of Science, Assist. Prof., 大学院・理学研究科, 助手 (70273430)
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| Project Period (FY) |
2001 – 2003
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| Project Status |
Completed (Fiscal Year 2003)
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| Budget Amount *help |
¥31,100,000 (Direct Cost: ¥31,100,000)
Fiscal Year 2003: ¥7,700,000 (Direct Cost: ¥7,700,000)
Fiscal Year 2002: ¥7,700,000 (Direct Cost: ¥7,700,000)
Fiscal Year 2001: ¥15,700,000 (Direct Cost: ¥15,700,000)
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| Keywords | oxygenase / reaction mechanism / non-heme iron / catechol 2,3-dioxygenase / catechol / oxygen activation / substituent effect / homoprotocatechuic acid / 結晶構造 / 2,3-ジヒドロキシビフェニール / 電荷移動錯体 / ニトロフェノール / 酵素添加酵素 / カテコール2,3-ジオキゲナーゼ / 酸素分子 |
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| Research Abstract |
1. Crystal structure of homoprotocatechuate 2,3-dioxygenase from Thermus thermophilus HB8 (HPCD) has been determined. Functional characterization of HPCD has been carried out. The enzyme shows unusual tolerance for hydrogen peroxide and a large K_m value for O_<2->
2. The kinetic parameters and substrate binding constants of catechol 2,3-dioxygenase (Mpc) and 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC) have been determined using improved assay methods. Comparison of the functional properties on the basis of their crystal structures has revealed that the interaction of C3-substituent group with the active site residues facilitates substrate binding in the case of BphC and substrate dissociation in the case of Mpc, respectively.
3. We have demonstrated negative correlation between the substrate binding rate of Mpc and electron-withdrawing nature of the substituent, steric effect of the substituent on the stability of the Mpc-substrate complex (EA complex), and positive correlation between
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the stability of Mpc-phenol complex (EI complex) and electron-withdrawing nature of the substituent. These results support a hypothesis that the binding of catechol proceeds through an intermediate resembling to EI complex in which catechol binds monodentately to the active site ferrous ion.
4. We have found negative correlation between the rate of oxygen binding to EA complex of Mpc and electron-withdrawing nature of the substituent. The result indicates that one-electron transfer from catechol to O_2 via the iron center activatesO_2 for the subsequent reactions. The oxygen binding site of Mpc is formed by the benzene ring of substrate, Hisl99, A202, Leu155, and Phe191. Leu155 is replaced by Val in that of BphC, and by Asn in that of HPCD, respectively.
5. We have found suicide-like inactivation of BphC by C4-substituted catechols. Crystal structure of the BphC-4-methylcatechol complex has revealed an aberrant bidentate binding mode different from that of 3-phenylcatechol.
In conclusion, in the present study, we have revealed structural and mechanistic basis of substrate recognition and O_2 activation. Directed evolution of extradiol dioxygenases is now possible based on rational design. Less
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