| Budget Amount *help |
¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2014: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000)
Fiscal Year 2013: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2012: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
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| Outline of Final Research Achievements |
An operon coding quinohemoprotein amine dehydrogenase (QHNDH) contains an ORF2 protein that generates three intra-peptidyl thioether crosslinks between Cys and Asp/Glu residues in the gamma subunit of QHNDH. As a result, 5-8 residues of loop regions were formed in the multi-loop structure of the gamma subunit. To elucidate the reaction mechanism of the protein and to construct peptide library cyclized by thioether bond formation, the ORF2 protein was purified under anaerobic conditions. Reconstitution of FeS cluster provided the active form catalyzing thioether bond formation in vitro. On the basis of the homology model of the ORF2 complexed with the gamma subunit, the details of the reaction mechanism were revealed in this study. In the short type gamma subunit containing a single crosslink site, it is successful that various sequences as well as randomized sites are introduced to the loop region.
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