| Co-Investigator(Kenkyū-buntansha) |
NAGAMURA Toshihiko YUNISOKU CO., DEVELOP.RESEARCH INST,DIRECTOR, 科学機器開発研究所, 所長
MIYOSHI Hideto THE UNIVERSITY OF TOKYO,FAC.OF AGRICULTURE,KYOTO UNIV., ASST PROFESSOR, 農学部, 助教授 (20190829)
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| Research Abstract |
The aims of this project are to develop new bacteriocides targetted to quinol oxidases of the aerobic respiratory chain and to elucidate biochemically the molecular mechanism of the substrate oxidation. Results obtained are follows ;
1. Based on structure-function studies on ubiquinone analogues, we found that the substrate oxidation sites (Q_I) of the Escherichia coli cytochromes bo and bd, which are not structurally related, have steric constraints against the 2-methoxy and 5-methyl groups. In addition, the 6-isoprenyl group increases the binding affinity of cytochrome bo.
2. Substrate specificity of the high affinity quinone binding site (Q_H) of cytochrome bo was different from that of the Q_L site. Reconstitution of the Q_H site with substituted phenols, flow-flash studies of the reduced Q_H-free enzyme with dioxygen and redox titrations indicated that the Q_H site mediates electron transfer from the Q_L site to low-spin heme b.
3. Screening of antimycin A,rorenone and capsaicin derivatives revealed that some antimycin A derivatives can inhibit quinol oxidases strongly.
4. Among new quinone analogues identified in this study, three compounds were shown to inhibit the aerobic growth of E.coli and can be used as a lead compound for design of terminal oxidase-specific bacteriocides. Then, we isolated and characterized the quinone analogue-resistant mutants of cytochrome bo biochemically and molecular biologically. Results indicate that two quinone/quinol binding sites in cytochrome bo function cooperatively to catalyze the quinol oxidation and electron transfer from the Q_L to heme b in subunit I.
5. Molecular biological and chemical cross-linking studies indicate that the structure of cytochrome bo is similar to that of cytohcrome c oxidase from Paracoccus denitrificans. To determine an atomic structure of cytochrome bo, the examination of the crystallization conditions is in progress.
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