2018 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 mechanism / sequence evolution / kinetic isotope effect / sulfate reduction / geobiology |
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| Outline of Annual Research Achievements |
In this year, we obtained the constructs for Apr enzyme homologs which will allow co-expression of the alpha and beta subunits from the same plasmids from multiple cloning sites. We also hired a new postdoctoral researcher, who is currently expressing and purifying these homologs. In addition, we developed a new protocol to synthesize the substrate of the enzyme (APS), using a polyphosphatase enzyme as a phosphorylating agent. The new system consumes product, and provides substrate, therefore we expect to be able to accurately measure burst phase kinetics in the absence of product inhibition.
In addition to starting characterization of these new enzyme homologs, we finished the characterization of Apr from the organism Desulfovibrio vulgaris Miyazaki strain F. These findings were published this year and form the foundation of our future comparisons.
We showed that the Apr enzyme may itself exert a regulatory influence on overall whole cell isotope fractionations, and integrated the KIE measurement of the enzyme into a steady state reaction model of cell physiology to interpret the results. Our findings indicate indicate that cells which have ample thermodynamic driving potential, and have sufficient sulfate as a respiratory substrate, display isotope fractionation values near the KIE value of the enzyme, thus creating a link between specific enzyme KIE and whole cell physiology.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
Our progress is proceeding as planned.
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| Strategy for Future Research Activity |
The plan for our future work remains the same as in the initial proposal.
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