2020 Fiscal Year Annual Research Report
Determining sulfur isotope fractionation values of individual enzymes and how they evolve through time.
| Project/Area Number |
18H01325
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| Research Institution | Tokyo Institute of Technology |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
上野 雄一郎 東京工業大学, 理学院, 教授 (90422542)
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | enzyme evolution / sulfur isotopes / metabolic flux / energy metabolism / geobiology / Earth history / history of life / enzyme kinetics |
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| Outline of Annual Research Achievements |
In this year, we studied the heterologous expression of ApsR enzymes in E.coli. We took two strategies. 1) the alpha and beta subunits were transcribed off plasmids with two multiple cloning sites and 2) linearized DNA from the enzymes was transcribed and translated in a cell free system. We worked with 20 ApsR homologs, which span most of the evolutionary variability of the enzyme family as viewed on a phylogenetic tree.
In (1), we found that the protein expresses poorly in E.coli. We varied cultivation and expression conditions extensively, yet we could not achieve good expression yields.
In (2), we identified a novel proteolytic cleavage of the protein occurs. The relationship of this cleavage to in-vivo activity is currently unknown, but it may be relevant to the operation of the protein. Further work is needed to evaluate this.
In addition to this laboratory work, we conducted bioinformatics analyses of the protein. First, we investigated the possible modes of product inhibition, which we observed and identified in our previous years research. From a structural perspective, we were able to rationalize how substrates or products may bind near the active site, affecting the reaction kinetics. From a structural and sequence evolution perspective, we were able to make progress in the understanding of the evolution of the family and structural domain (fold).
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| Research Progress Status |
令和2年度が最終年度であるため、記入しない。
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| Strategy for Future Research Activity |
令和2年度が最終年度であるため、記入しない。
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[Journal Article] Ten years of collaborative progress in the Quest for Orthologs2021
Author(s)
Linard Benjamin、Ebersberger Ingo、McGlynn Shawn E、Glover Natasha、Mochizuki Tomohiro、Patricio Mateus、Lecompte Odile、Nevers Yannis、Thomas Paul D、Gabaldon Toni、Sonnhammer Erik、Dessimoz Christophe、Uchiyama Ikuo、QFO Consortium
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Journal Title
Molecular Biology and Evolution
Volume: -
Pages: -
DOI
Peer Reviewed / Open Access / Int'l Joint Research
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